N-terminal epitopes in amyloid beta and conformationally-selective antibodies thereto

ABSTRACT

The disclosure pertains to N-terminal epitopes identified in A-beta, including conformational epitopes, antibodies thereto and methods of making and using immunogens and antibodies specific thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry of PCT/CA2016/051300, filedNov. 9, 2016, which claims priority from U.S. Provisional patentapplication Ser. Nos. 62/253,044 filed Nov. 9, 2015; 62/331,925 filedMay 4, 2016; 62/365,634 filed Jul. 22, 2016; and 62/393,615 filed Sep.12, 2016; each of these applications being incorporated herein in theirentirety by reference.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“P50440US01_SequenceListing.txt” (7,668 bytes), submitted via EFS-WEBand created on May 9, 2018, is herein incorporated by reference.

FIELD

The present disclosure relates to N-terminal Amyloid beta (A-beta or Aβ)epitopes and antibodies thereto and more specifically to conformationalA-beta epitopes that are for example selectively accessible in A-betaoligomers, and related antibody compositions.

BACKGROUND

Amyloid-beta (A-beta), which exists as a 36-43 amino acid peptide, is aproduct released from amyloid precursor protein (APP) by the enzymes βand γ secretase. In Alzheimer's disease (AD) patients, A-beta can bepresent in soluble monomers, insoluble fibrils and soluble oligomers. Inmonomer form, A-beta exists as a predominantly unstructured polypeptidechain. In fibril form, A-beta can aggregate into distinct morphologies,often referred to as strains. Several of these structures have beendetermined by solid-state NMR.

For, example, structures for several strains of fibrils are available inthe Protein Data Bank (PDB), a crystallographic database of atomicresolution three dimensional structural data, including a 3-foldsymmetric Aβ structure (PDB entry, 2M4J); a two-fold symmetric structureof Aβ-40 monomers (PDB entry 2LMN), and a single-chain, parallelin-register structure of Aβ-42 monomers (PDB entry 2MXU).

The structure of 2M4J is reported in Lu et al [8], and the structure of2MXU is reported in Xiao et al [9]. The structure of 2LMN is reported inPetkova et al [10].

A-beta oligomers have been shown to kill cell lines and neurons inculture and block a critical synaptic activity that subserves memory,referred to as long term potentiation (LTP), in brain slice cultures andliving animals.

The structure of the oligomer has not been determined to date. Moreover,NMR and other evidence indicates that the oligomer exists not in asingle well-defined structure, but in a conformationally-plastic,malleable structural ensemble with limited regularity. Moreover, theconcentration of toxic oligomer species is far below either that of themonomer or fibril (estimates vary but are on the order of 1000-foldbelow or more), making this target elusive.

Antibodies that bind A-beta have been described.

U.S. Pat. No. 7,780,963 Anti-ADDL Antibodies relates to antibodies thatdifferentially recognize multidimensional conformations of A-betaderived diffusible ligands

U.S. Pat. No. 9,176,151 describes selective anti-Aβ oligomer antibodies,kits and an immunoassay method using a pair of anti-Aβ oligomerantibodies for detecting Aβ oligomers in a biological sample of apatient.

WO2003070760 titled ANTI-AMYLOID BETA ANTIBODIES AND THEIR USE isdirected towards antibody molecules capable of specifically recognizingtwo regions of the β-A4 peptide, wherein the first region comprises theamino acid sequence AEFRHDSGY or a fragment thereof and wherein thesecond region comprises the amino acid sequence VHHQKLVFFAEDVG or afragment thereof.

WO2006066089 titled HUMANIZED AMYLOID BETA ANTIBODIES FOR USE INIMPROVING COGNITION is directed to improved agents and methods fortreatment of diseases associated with beta amyloid and in particular tothe identification and characterization of a monoclonal antibody, 12A11,that specifically binds to Aβ peptide and is effective at reducingplaque burden associated with amyloidogenic disorders (e.g., AD).

WO2007068429 titled ANTIBODIES AGAINST AMYLOID BETA 4 WITH GLYCOSYLATEDIN THE VARIABLE REGION is directed to a purified antibody moleculepreparation being characterized in that at least one antigen bindingsite comprises a glycosylated asparagine (Asn) in the variable region ofthe heavy chain (V_(H)).

Yu et al. describes a hexavalent foldable Aβ1-15 (6Aβ5) fused to PADREor toxin-derived carrier proteins. Wang et al. 2016 report thatperipheral administration of this antibody mitigates Alzheimer's diseaselike pathology and cognitive decline in a transgenic animal model ofaged Alzheimer Disease [11], [12].

Antibodies that preferentially or selectively bind A-beta oligomers overmonomers or over fibrils or over both monomers and fibrils aredesirable.

SUMMARY

Described herein are epitopes and more particularly conformationalepitopes, in A-beta comprising and/or consisting of residues HDSG (SEQID NO:1) or related epitopes, and antibodies that specifically and/orselectively bind said epitopes. The epitopes may be selectively exposedin the oligomeric species of A-beta, in a conformation thatdistinguishes oligomeric species from that in the monomer and/or fibril.

An aspect includes a cyclic compound, preferably a cyclic compound,comprising: an A-beta peptide the peptide comprising HDS and up to 6A-beta contiguous residues, and a linker, wherein the linker iscovalently coupled to the A-beta peptide N-terminus residue and theA-beta C-terminus residue.

In an embodiment, the A-beta peptide is selected from a peptide having asequence of any one of SEQ ID NOS: 1-16, optionally selected from HDSG(SEQ ID NO: 1), HDSGY (SEQ ID NO: 4), HDSGYE (SEQ ID NO: 11), RHDSGY(SEQ ID NO: 13), RHDSG (SEQ ID NO: 5), RHDS (SEQ ID NO: 6) and DSGY (SEQID NO: 14).

In another embodiment, the cyclic compound is a cyclic peptide.

In another embodiment, the cyclic compound described herein comprises i)a curvature increase of D and/or S in the compound of at least 10%, atleast 20%, or at least 30% compared to D or S in the context of acorresponding linear compound; ii) at least one residue selected from H,D and S, wherein at least one dihedral angle of said residue isdifferent by at least 30 degrees, at least 40 degrees, at least 50degrees, at least 60 degrees, at least 70 degrees, or at least 80degrees compared to the corresponding dihedral angle in the context of acorresponding linear compound; iii) an O-C-Cα-Cβ dihedral angle in Dthat is different by at least 30 degrees, at least 40 degrees, at least50 degrees, at least 60 degrees, at least 70 degrees or at least 80degrees compared to the corresponding dihedral angle in the context of acorresponding linear compound; and/or iv) a conformation for H and/or Das measured by entropy that is at least 10%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40% more constrained compared to acorresponding linear compound.

In another embodiment, the A-beta peptide is selected from HDSG (SEQ IDNO: 1), HDSGY (SEQ ID NO: 4) and RHDSG (SEQ ID NO: 5).

In another embodiment, the compound further comprises a detectablelabel.

In another embodiment, the linker comprises or consists of 1-8 aminoacids and/or equivalently functioning molecules and/or one or morefunctionalizable moieties.

In another embodiment, the linker amino acids are selected from A and G,and/or wherein the functionalizable moiety is C.

In another embodiment, the linker comprises or consists of amino acidsGCG or CGC.

In another embodiment, the linker comprises a PEG molecule.

In another embodiment, the cyclic compound is selected from thestructures shown in FIG. 11B.

An aspect includes an immunogen comprising the cyclic compound describedherein.

In an embodiment, the compound is coupled to a carrier protein orimmunogenicity enhancing agent.

In another embodiment, the carrier protein is bovine serum albumin (BSA)or the immunogenicity-enhancing agent is keyhole Keyhole LimpetHaemocyanin (KLH).

An aspect includes a composition comprising the compound describedherein or the immunogen described herein.

In an embodiment, the immunogen comprising compositions described hereinfurther comprises an adjuvant.

In another embodiment, the adjuvant is aluminum phosphate or aluminumhydroxide.

An aspect includes an isolated antibody that specifically binds to anA-beta peptide having a sequence of HDSG (SEQ ID NO: 1) or a relatedepitope sequence, optionally as set forth in any one of SEQ ID NOS:1-16.

In an embodiment, the antibody specifically binds an epitope on A-beta,wherein the epitope comprises at least two consecutive amino acidresidues predominantly involved in binding to the antibody, wherein theat least two consecutive amino acids are DS embedded within HDS, orwherein the at least two consecutive amino acids are HD embedded withinHDS.

In another embodiment, the epitope comprises or consists of HDS, DSG,HDSG (SEQ ID NO: 1), HDSGY (SEQ ID NO: 4), HDSGYE (SEQ ID NO: 11),RHDSGY (SEQ ID NO: 13), RHDSG (SEQ ID NO: 5), RHDS (SEQ ID NO: 6) andDSGY (SEQ ID NO: 14).

In another embodiment, the antibody is a conformation specific and/orselective antibody that specifically or selectively binds to HDSG (SEQID NO: 1) or a related epitope peptide presented in a cyclic compound,optionally a cyclic compound described herein, preferably a cyclicpeptide having a sequence as set forth in SEQ ID NO: 2 or 12.

In another embodiment, the antibody selectively binds A-beta oligomerover A-beta monomer and/or A-beta fibril.

In another embodiment, the selectivity is at least 2 fold, at least 3fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30fold, at least 40 fold, at least 50 fold, at least 100 fold, at least500 fold, at least 1000 fold more selective for A-beta oligomer overA-beta monomer and/or A-beta fibril.

In another embodiment, the antibody does not specifically and/orselectively bind a linear peptide comprising sequence HDSG (SEQ IDNO: 1) or a related epitope, optionally wherein the sequence of thelinear peptide is a linear version of a cyclic compound used to raisethe antibody, optionally a linear peptide having a sequence as set forthin SEQ ID NO: 2 or 12.

In another embodiment, the antibody lacks or has negligible binding toA-beta monomer and/or A-beta fibril plaques in situ.

In another embodiment, the antibody is a monoclonal antibody or apolyclonal antibody.

In another embodiment, the antibody is a humanized antibody.

In another embodiment, the antibody is an antibody binding fragmentselected from Fab, Fab′, F(ab′)₂, scFv, dsFv, ds-scFv, dimers,nanobodies, minibodies, diabodies, and multimers thereof.

Another embodiment comprises a light chain variable region and a heavychain variable region, optionally fused, the heavy chain variable regioncomprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, the light chain variable region comprising complementaritydetermining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acidsequences of said CDRs comprising the sequences:

(SEQ ID NO: 17) CDR-H1 GYTFTSYW (SEQ ID NO: 18) CDR-H2 IDPSDSQT(SEQ ID NO: 19) CDR-H3 SRGGY (SEQ ID NO: 20) CDR-L1 QDINNY(SEQ ID NO: 21) CDR-L2 YTS (SEQ ID NO: 22) CDR-L3 LQYDNLWT

In another embodiment, the antibody comprises a heavy chain variableregion comprising: i) an amino acid sequence as set forth in SEQ ID NO:24; ii) an amino acid sequence with at least 50%, at least 60%, at least70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 24,wherein the CDR sequences are as set forth in SEQ ID NO: 17, 18 and 19,or iii) a conservatively substituted amino acid sequence i).

In another embodiment, the antibody comprises a light chain variableregion comprising i) an amino acid sequence as set forth in SEQ ID NO:26, ii) an amino acid sequence with at least 50%, at least 60%, at least70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 26,wherein the CDR sequences are as set forth in SEQ ID NO: 20, 21 and 22,or iii) a conservatively substituted amino acid sequence of i).

In another embodiment, the heavy chain variable region amino acidsequence is encoded by a nucleotide sequence as set forth in SEQ ID NO:23 or a codon degenerate or optimized version thereof; and/or theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 25 or a codondegenerate or optimized version thereof.

In another embodiment, the heavy chain variable region comprises orconsists of an amino acid sequence as set forth in SEQ ID NO: 24 and/orthe light chain variable region comprises or consists of an amino acidsequence as set forth in SEQ ID NO: 26.

In another embodiment, the antibody competes for binding to human A-betawith an antibody comprising the CDR sequences as recited in Table 10.

In an embodiment, the antibody is prepared using a cyclic compound orimmunogen described herein.

An aspect includes an immunoconjugate comprising an antibody describedherein and a detectable label or cytotoxic agent.

In an embodiment, the detectable label comprises a positron emittingradionuclide, optionally for use in subject imaging such as PET imaging.

An aspect includes an antibody described herein, or an immunoconjugatedescribed herein, optionally with a diluent.

An aspect includes a nucleic acid molecule encoding a proteinaceousportion of the compound or immunogen described herein, the antibodydescribed herein or proteinaceous immunoconjugates described herein.

An aspect includes a vector described herein.

An aspect includes a cell expressing an antibody described herein,optionally wherein the cell is a hybridoma comprising the vector.

An aspect includes a kit described herein, the immunogen describedherein, the antibody described herein, the immunoconjugate describedherein, the nucleic acid molecule described herein, the vector describedherein or the cell described herein.

An aspect includes a method of making the antibody described herein,comprising administering the compound or immunogen described herein or acomposition comprising said compound or immunogen to a subject andisolating antibody and/or cells expressing antibody specific orselective for the compound or immunogen administered and/or A-betaoligomers, optionally lacking or having negligible binding to a linearpeptide comprising the A-beta peptide and/or lacking or havingnegligible plaque binding.

An aspect includes a method of determining if a biological samplecomprises A-beta, the method comprising:

-   -   a. contacting the biological sample with an antibody described        herein or the immunoconjugate described herein; and    -   b. detecting the presence of any antibody complex.

In an embodiment, the method described herein for determining if thebiological sample contains A-beta oligomer the method comprising:

-   -   a. contacting the sample with the antibody described herein or        the immunoconjugate described herein that is specific and/or        selective for A-beta oligomers under conditions permissive for        forming an antibody: A-beta oligomer complex; and    -   b. detecting the presence of any complex;

wherein the presence of detectable complex is indicative that the samplemay contain A-beta oligomer.

In another embodiment, the amount of complex is measured.

In another embodiment, the sample comprises brain tissue or an extractthereof, whole blood, plasma, serum and/or CSF.

In another embodiment, the sample is a human sample.

In another embodiment, the sample is compared to a control, optionally aprevious sample.

In another embodiment, the level of A-beta is detected by SPR.

An aspect includes a method of measuring a level of A-beta in a subject,the method comprising administering to a subject at risk or suspected ofhaving or having AD, an immunoconjugate comprising an antibody describedherein wherein the antibody is conjugated to a detectable label; anddetecting the label, optionally quantitatively detecting the label.

In an embodiment the label is a positron emitting radionuclide.

An aspect includes a method of inducing an immune response in a subject,comprising administering to the subject a compound or combination ofcompounds described herein, optionally a cyclic compound comprising HDSG(SEQ ID NO: 1) or a related epitope peptide sequence, an immunogenand/or composition comprising said compound or said immunogen; andoptionally isolating cells and/or antibodies that specifically orselectively bind the A-beta peptide in the compound or immunogenadministered.

An aspect includes a method of inhibiting A-beta oligomer propagation,the method comprising contacting a cell or tissue expressing A-beta withor administering to a subject in need thereof an effective amount of anA-beta oligomer specific or selective antibody or immunoconjugatedescribed herein, to inhibit A-beta aggregation and/or oligomerpropagation.

An aspect includes a method of treating AD and/or other A-beta amyloidrelated diseases, the method comprising administering to a subject inneed thereof i) an effective amount of an antibody or immunoconjugatedescribed herein, optionally an A-beta oligomer specific or selectiveantibody, or a pharmaceutical composition comprising said antibody; 2)administering an isolated cyclic compound comprising HDSG (SEQ ID NO: 1)or a related epitope sequence or immunogen or pharmaceutical compositioncomprising said cyclic compound, or 3) a nucleic acid or vectorcomprising a nucleic acid encoding the antibody of 1 or the immunogen of2, to a subject in need thereof.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein.

In an embodiment, more than one antibody or immunogen is administered.

In an embodiment, the antibody, immunoconjugate, immunogen, compositionor nucleic acid or vector is administered directly to the brain or otherportion of the CNS.

In an embodiment, the composition is a pharmaceutical compositioncomprising the compound or immunogen in admixture with apharmaceutically acceptable, diluent or carrier.

An aspect includes an isolated peptide comprising an A beta peptideconsisting of the sequence of any one of the sequences set forth in SEQID NOS: 1-16.

In an embodiment, the isolated peptide is a cyclic peptide comprising alinker wherein the linker is covalently coupled to the A-beta peptideN-terminus residue and/or the A-beta C-terminus residue.

In an embodiment, the isolated cyclic peptide comprises a detectablelabel.

An aspect includes a nucleic acid sequence encoding the isolated peptidedescribed herein.

An aspect includes a hybridoma expressing the antibody described herein.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1: Likelihood of exposure as a function of sequence, as determinedby the collective coordinates method.

FIG. 2: Curvature as a function of residue index. Mean curvature in theequilibrium ensemble for the cyclic peptide CGHDSGG (SEQ ID No: 2) isshown (solid light grey), along with the curvature for the linearpeptide (solid dark grey), and the curvature of the various monomers inthe fibril (dotted line).

FIG. 3: Dihedral angle distributions for the side chain heavy atoms ofH6. Schematics of residue H6 are shown in the insets; the correspondingbond over which the dihedral angle is taken is rendered darker than theother bonds, and the four atoms defining the dihedral angle are shown inlighter gray. The angles corresponding to the peak values of thedihedral distributions for all 3 species-linear peptide, cyclic peptide,and (2M4J) fibril ensemble are provided in Table 1. The differencesbetween the peak values are also provided in Table 1. The dihedral angledistribution for the fibril ensemble is taken over all 9 chains ofA-beta42 in the PDB structure in the fibril, so the dihedraldistribution observed is generally broader than the distribution for anysingle chain taken from the structure 2M4J.

FIG. 4: Dihedral angle distribution for the angle O-C-Cα-Cβ involvingthe side chain heavy atoms of residue D7. Schematics of D7 are shown inthe insets; the corresponding bond over which the dihedral angle istaken is rendered darker than the other bonds. The values are providedin Table 1.

FIG. 5: Dihedral angle distributions for angles involving the side chainheavy atoms of S8. Schematics of S8 are shown in the insets; thecorresponding bond over which the dihedral angle is taken is rendereddarker than the other bonds. The values are provided in Table 1.

FIG. 6: Top panel (A): Side chain entropy change of the linear andcyclic peptides relative to the entropy in the fibril, plotted for eachresidue H, D, and S. (B) 2^(nd) from top panel: entropy of theindividual dihedral angles in H6. Note for example that CA-CB-CG-ND1 hassubstantially less entropy than either the fibril or linear peptides,which can also be seen by the more sharply peaked dihedral angledistribution for this particular dihedral in FIG. 3. (C) 2^(nd) frombottom panel: entropy of individual dihedral angles in D7. (D) Bottompanel: entropy of individual dihedral angles in S8.

FIG. 7: Equilibrium backbone Ramachandran angles for residues H, D, S,and G, in both the linear and cyclic forms of the peptide CGHDSGG (SEQID NO: 2), along with the backbone Ramachandran angles for the residuesH, D, S, and G in the context of the fibril 2M4J.

FIG. 8: Solubility vs residue index for A-beta42 peptide. HDSG (SEQ IDNO: 1) has values of +1.1, +0.14, +1.2, and +0.30 respectively.

FIG. 9: Plots of the solvent accessible surface area (SASA), theweighted SASA, ((s, −<s>)/δs) SASA, and ((s, −<s>)/δs)·SASA,−(((s_(i)−<s>)/δs)·SASA_(i))_(fibril).

FIG. 10: Two separate views of the root mean squared deviation (RMSD)values to the centroids of the three largest clusters of the linearpeptide ensemble. Each point corresponds to a given conformation takenfrom the linear peptide, cyclic peptide, or fibril equilibriumensembles.

FIG. 11A: Two views of the cyclic peptide structure CGHDSGG (SEQ ID NO:2), rendered in licorice representation so the orientations of the sidechains can be seen. The light gray colored conformation is the centroidof the largest cluster, as described above for FIG. 10, and bestrepresents the typical conformation of the cyclic peptide. The blackside chains are rendered for a linear conformation having dihedralangles close to the most likely dihedral angles of the linear peptide;the side chains for this linear peptide conformation are superimposed onthe cyclic peptide, to show that different dihedral angles tend to bepreferred for the linear and cyclic peptides.

FIG. 11B: Schematic representations of cyclic peptides comprising HDSG(SEQ ID NO: 1), including the cyclic peptide with circular peptide bond,the cyclic peptide with PEG2 linker between the G and C residues, andthe cyclic peptide with PEG2 linker between the C and H residues.

FIG. 12: Clustering plots by root mean squared deviation (RMSD); axescorrespond again to the centroids of the three largest clusters of thelinear peptide ensemble, as in FIG. 10.

FIG. 13: Surface plasmon resonance (SPR) direct binding assay ofantibodies to cyclic peptide and linear peptide in Panel A, and A-betaoligomer and A-beta monomer in Panel B.

FIG. 14: Primary screening of clones from tissue culture supernatantsusing ELISA and SPR direct binding assay. Plot comparing mAb binding inSPR direct binding assay versus ELISA.

FIG. 15: SPR direct binding assay of select clones to cyclic peptide(structured peptide: circles), linear peptide (unstructured peptide,squares), A-beta monomer (upward pointing triangles), and A-betaoligomer (downward pointing triangles). Asterisk indicates a clonereactive to unstructured linear peptide for control purposes.

FIG. 16: Immunohistochemical staining of plaque from cadaveric AD brainusing 6E10 positive control antibody (A) and an antibody (303-25-1B4)raised against cyclo(CGHDSGG) (SEQ ID NO:2) (B).

FIG. 17: Secondary Screening of selected and purified antibodies usingan SPR indirect (capture) binding assay. SPR binding response of A-betaoligomer to captured antibody minus binding response of A-beta monomerto captured antibody (circle); SPR binding response of pooled solublebrain extract from AD patients to captured antibody minus bindingresponse of pooled brain extract from non-AD controls to capturedantibody (triangle); SPR binding response of pooled cerebrospinal fluid(CSF) from AD patients to captured antibody minus binding response ofpooled CSF from non-AD controls to captured antibody (square).

FIG. 18: Verification of Antibody binding to A-beta oligomers. SPRsensorgrams and binding response plots of varying concentrations ofcommercially-prepared stable A-beta oligomers binding to immobilizedantibodies. Panel A shows results with the positive control mAb6E10,Panel B with the negative isotype control and Panel C with antibodyraised against cyclo (CGHDSGG) (SEQ ID NO:2). Panel D plots binding ofseveral antibody clones raised against cyclic peptide comprising HDSG(SEQ ID No: 1), with A-beta oligomer at a concentration of 1 micromolar.

FIG. 19: A plot showing propagation of A-beta aggregation in vitro inthe presence or absence of a representative antibody raised using acyclic peptide comprising HDSG (SEQ ID NO:1).

Table 1 shows the peak values of the dihedral angle distribution forthose dihedral angles whose distributions show significant differencesbetween the cyclic peptide and other species.

Table 2 shows peak values of the Ramachandran backbone phi/psi angledistributions.

Table 3 gives the Ramachandran backbone dihedral angles as well as theside chain dihedral angles for the cyclic peptide that is the centroidconformation of the largest conformational cluster, and for the centroidconformation of the largest cluster taken from the linear peptideensemble.

Table 4 is a table of mean curvature values for each residue in thecyclic, linear, and 2M4J fibril ensembles.

Table 5 shows the binding properties of selected antibodies.

Table 6 shows the binding properties summary for selected antibodies.

Table 7 lists the oligomer binding—monomer binding for an antibodyraised against cyclo(CGHDSGG) (SEQ ID NO:2).

Table 8 lists properties of antibodies tested on formalin fixed tissues.

Table 9 is an exemplary toxicity assay.

Table 10 lists CDR sequences.

Table 11 lists heavy chain and light chain variable sequences.

Table 12 is a table of A-beta epitope sequences and select sequenceswith linker.

Table 13 provides the full A-beta 1-42 human polypeptide sequence.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are antibodies, immunotherapeutic compositions andmethods which may target epitopes preferentially accessible in toxicoligomeric species of A-beta, including oligomeric species associatedwith Alzheimer's disease. A region in A-beta has been identified thatmay be specifically and/or selectively accessible to antibody binding inoligomeric species of A-beta.

As demonstrated herein, generation of oligomer-specific or oligomerselective antibodies was accomplished through the identification oftargets on A-beta peptide that are not present, or present to a lesserdegree, on either the monomer and/or fibril. Oligomer-specific epitopesneed not differ in primary sequence from the corresponding segment inthe monomer or fibril, however they would be conformationally distinctin the context of the oligomer. That is, they would present a distinctconformation in terms of backbone and/or side-chain orientation in theoligomer that would not be present (or would be unfavourable) in themonomer and/or fibril.

Antibodies raised to linear peptide regions may not to be selective foroligomer, and thus may bind to monomer or A-beta plaques as well.

As described herein, to develop antibodies that may be selective foroligomeric forms of A-beta, the inventors sought to identify regions ofA-beta sequence that are prone to disruption in the context of thefibril, and that may be exposed on the surface of the oligomer.

As described the Examples, the inventors have identified a region theyhave determined to be prone to disruption in the context of the fibril.The inventors designed cyclic compounds comprising the identified targetregion to satisfy criteria of an alternate conformation such as highercurvature, higher exposed surface area, alternative dihedral angledistributions, and/or did not readily align by root mean squareddeviation (RMSD) to either the linear or fibril ensembles.

Antibodies could be raised using a cyclic peptide comprising the targetregion, that selectively bound the cyclic peptide compared to a linearpeptide of the same sequence (e.g. corresponding linear sequence).Experimental results are described and identify epitope-specific andconformationally selective antibodies that bind synthetic oligomerselectively compared to synthetic monomers, bind CSF from AD patientspreferentially over control CSF and/or bind soluble brain extract fromAD patients preferentially over control soluble brain extract. Furtherstaining of AD brain tissue identified antibodies that show no ornegligible plaque binding and in vitro studies found that the antibodiesinhibited Aβ oligomer propagation and aggregation.

I. Definitions

As used herein, the term ‘A-beta’ may alternately be referred to as‘amyloid beta’, ‘amyloid 13’, Abeta, A-beta or A13′. Amyloid beta is apeptide of 36-43 amino acids and includes all wildtype and mutant formsof all species, particularly human A-beta. A-beta40 refers to the 40amino acid form; A-beta42 refers to the 42 amino acid form, etc. Theamino acid sequence of human wildtype A-beta42 is shown in SEQ ID NO: 3

As used herein, the term “A-beta monomer” herein refers to any of theindividual subunit forms of the A-beta (e.g. 1-40, 1-42, 1-43) peptide.

As used herein, the term “A-beta oligomer” herein refers to a pluralityof any of the A-beta subunits wherein several (e.g. at least two) A-betamonomers are non-covalently aggregated in a conformationally-flexible,partially-ordered, three-dimensional globule of less than about 100, ormore typically less than about 50 monomers. For example, an oligomer maycontain 3 or 4 or 5 or more monomers. The term “A-beta oligomer” as usedherein includes both synthetic A-beta oligomer and/or native A-betaoligomer. “Native A-beta oligomer” refers to A-beta oligomer formed invivo, for example in the brain and CSF of a subject with AD.

As used herein, the term “A-beta fibril” refers to a molecular structurethat comprises assemblies of non-covalently associated, individualA-beta peptides which show fibrillar structure under an electronmicroscope. The fibrillar structure is typically a “cross beta”structure; there is no theoretical upper limit on the size of multimers,and fibrils may comprise thousands or many thousands of monomers.Fibrils can aggregate by the thousands to form senile plaques, one ofthe primary pathological morphologies diagnostic of AD.

The term “HDSG” means the amino acid sequence histidine, aspartic acid,serine, and glycine as shown in SEQ ID NO: 1. Similarly DSG, DSGG (SEQID NO:3), HDSGYE (SEQ ID NO:11), HDSGY (SEQ ID NO:4), RHDSG (SEQ IDNO:5), RHDS (SEQ ID NO:6) refer to the amino acid sequence identified bythe 1-letter amino acid code. Depending on the context, the reference ofthe amino acid sequence can refer to a sequence in A-beta or an isolatedpeptide, such as the amino acid sequence of a cyclic compound.

The term “alternate conformation than occupied by an amino acid residue(e.g. H, D, S and/or G) in the linear compound, monomer and/or fibril”as used herein means having one or more differing conformationalproperties selected from solvent accessibility, entropy, curvature (e.g.in the context of peptide HDSG (SEQ ID NO:1) as compared to for examplein the cyclic peptide described in the Examples), RMSD structuralalignment, and dihedral angle of one or more backbone or side chaindihedral angles compared to said property for H, D and/or S in an A-betalinear compound comprising the residue in context, A-beta monomer and/orA-beta fibril structures as shown for example in PDBs 2M4J, 2MXU, 2LMN,or 2LMP and shown in FIGS. 1-12 and/or in the Tables. For example, FIG.2 and Table 4 show that the curvature of HDSG (SEQ ID NO:1), for thecyclic peptide ensemble is significantly larger than the curvature ofHDSG (SEQ ID NO:1), in the ensemble of fibril conformations. This isparticularly evident for D7, S8, and G9. Moreover for D7 and S8, thecurvature in the cyclic peptide ensemble is substantially higher thanthat in the linear peptide ensemble. This implies conformationalselectivity may be particularly conferred by residues D7 and S8. Thelast two panels of FIG. 3 show that the dihedral angle distribution forthe angles (N-CA-CB-CG) and (C-CA-CB-CG) for H6 in the cyclic peptideensemble do have overlap, but are not the most common angles in thelinear peptide and fibril ensembles (the probabilities are 36% and 13%respectively for N-CA-CB-CG in linear and fibril and 36% and 13%respectively for C-CA-CB-CG in the linear and fibril). The last panel ofFIG. 4 shows that the dihedral angle distribution for angle (O-C-CA-CB)involving the side chain of residue D7 reflects an alternateconformational distribution compared to either the monomer or fibril.FIG. 5 shows that the dihedral angle distributions for angles(N-CA-CB-OG), (C-CA-CB-OG), and (O-C-CA-CB) involving the side chain ofresidue S8 reflects an alternate conformational distribution compared toeither the monomer or fibril. The alternate conformation can besimilarly, less or more “constrained” than the comparator conformation.For example, FIG. 6 demonstrates that H6 is more constrained in thecyclic peptide then it is in either the fibril or the monomer. ResidueD7 is more constrained in the cyclic peptide ensemble then it is in themonomer, but less than it is in the fibril. Residue S8 is lessconstrained in the cyclic peptide ensemble then it is in the fibril andalso marginally less than it is in the monomer. FIG. 7 demonstrates thatthe distributions of the Ramachandran dihedral angles for the backboneof cyclic peptides are substantially different than those for eithermonomer or fibril for residues D7 and S8. FIG. 8 shows that the residuesHDSG (SEQ ID NO:1) have a larger solubility than the average solubilitypresent in A-beta peptide, indicating the likelihood of exposure ofthese residues in an oligomeric ensemble of conformations. FIG. 9 showsthat residues HDSG (SEQ ID NO:1) have increased solvent accessiblesurface area, SASA, compared to the fibril, and that, when weighted bysolubility, all residues in the cyclic peptide ensemble show an increasein weighted SASA over that in the fibril, with residue S8 showingsubstantial increase in weighted SASA over the fibril. FIG. 10 showsthat the cyclic peptide equilibrium structures of HDSG (SEQ ID NO:1)cluster differently than the equilibrium structures of either the linearpeptide or corresponding sequence in the fibril, while the linear andfibril ensembles are not clearly differentiated.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified L-amino acids. The atoms of the amino acid caninclude different isotopes. For example, the amino acids can comprisedeuterium substituted for hydrogen nitrogen-15 substituted fornitrogen-14, and carbon-13 substituted for carbon-12 and other similarchanges.

The term “antibody” as used herein is intended to include, monoclonalantibodies, polyclonal antibodies, single chain, veneered, humanized andother chimeric antibodies and binding fragments thereof, including forexample a single chain Fab fragment, Fab′2 fragment or single chain Fvfragment. The antibody may be from recombinant sources and/or producedin animals such as rabbits, llamas, sharks etc. Also included are humanantibodies that can be produced in transgenic animals or usingbiochemical techniques or can be isolated from a library such as a phagelibrary. Humanized or other chimeric antibodies may include sequencesfrom one or more than one isotype or class or species.

The phrase “isolated antibody” refers to antibody produced in vivo or invitro that has been removed from the source that produced the antibody,for example, an animal, hybridoma or other cell line (such asrecombinant insect, yeast or bacteria cells that produce antibody). Theisolated antibody is optionally “purified”, which means at least: 80%,85%, 90%, 95%, 98% or 99% purity.

The term “binding fragment” as used herein to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain and which binds theantigen or competes with intact antibody. Exemplary binding fragmentsinclude without limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv,dimers, nanobodies, minibodies, diabodies, and multimers thereof.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. For example, F(ab′)2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab′)2fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Papain digestion can lead to the formation of Fab fragments.Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also beconstructed by recombinant expression techniques.

The terms “IMGT numbering” or “ImMunoGeneTics database numbering”, whichare recognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or antigen binding portion thereof.

When an antibody is said to specifically bind to an epitope such as HDSG(SEQ ID NO:1), what is meant is that the antibody specifically binds toa peptide containing the specified residues or a part thereof forexample at least 2 residues of HDSG, with a minimum affinity, and doesnot bind an unrelated sequence or unrelated sequence spatial orientationgreater than for example an isotype control antibody. Such an antibodydoes not necessarily contact each residue of HDSG (SEQ ID NO:1) andevery single amino acid substitution or deletion within said epitopedoes not necessarily significantly affect and/or equally affect bindingaffinity.

When an antibody is said to selectively bind an epitope such as aconformational epitope, such as HDSG (SEQ ID NO:1), what is meant isthat the antibody preferentially binds one or more particularconformations containing the specified residues or a part thereof withgreater affinity than it binds said residues in another conformation.For example, when an antibody is said to selectively bind a cyclopeptidecomprising HDSG or related epitope relative to a corresponding linearpeptide, the antibody binds the cyclopeptide with at least a 2 foldgreater affinity than it binds the linear peptide.

As used herein, the term “conformational epitope” refers to an epitopewhere the epitope amino acid sequence has a particular three-dimensionalstructure wherein at least an aspect of the three-dimensional structurenot present or less likely to be present in a corresponding linearpeptide is specifically and/or selectively recognized by the cognateantibody. The epitope e.g. HDSG (SEQ ID NO: 1) may be partially orcompletely exposed on the molecular surface of oligomeric A-beta andpartially or completely obscured from antibody recognition in monomericor fibrillar plaque A-beta. Antibodies which specifically and/orselectively bind a conformation-specific epitope recognize the spatialarrangement of one or more of the amino acids of thatconformation-specific/selective epitope. For example an HDSG (SEQ IDNO: 1) conformational epitope, refers to an epitope of HDSG (SEQ IDNO: 1) that is recognized by antibodies specifically and/or selectively,for example at least 2 fold, 3 fold, 5 fold, 10 fold, 50 fold, 100 fold,250 fold, 500 fold or 1000 fold or greater, more selectively as comparedto linear HDSG (SEQ ID NO: 1).

The term “related epitope” as used herein means at least two residues ofHDSG (SEQ ID NO:1) that are antigenic and/or sequences comprising 1 or 2amino acid residues in a A-beta either N-terminal or C-terminal to atleast two residues of HDSG (SEQ ID NO: 1). For example it is shownherein HDSG (SEQ ID NO:1), HDSGY (SEQ ID NO:4) and RHDSG (SEQ ID NO:5)were identified as regions prone to disorder in an A-beta fibril. HDSGY(SEQ ID NO:4) and RHDSG (SEQ ID NO:5) are accordingly related epitopes.Further it is demonstrated through modelling that residues D7 and S8 inparticular exhibit differences in the cyclic compound compared to thecorresponding linear sequence, accordingly DS, HDS, DSG, DSGY (SEQ IDNO: 13) and RHDS (SEQ ID NO:6) are related epitopes. Exemplary relatedepitopes can include A-beta sequences included in Table 12.

The term “constrained conformation” as used herein with respect to anamino acid or a side chain thereof, within a sequence of amino acids(e.g. H or D in HDSG (SEQ ID NO: 1)), or with respect to a sequence ofamino acids in a larger polypeptide, means decreased rotational mobilityof the amino acid dihedral angles, relative to a corresponding linearpeptide sequence, or the sequence or larger polypeptide, resulting in adecrease in the number of permissible conformations. This can bequantified for example by finding the entropy reduction for the ensembleof side chain dihedral angle degrees of freedom, and is plotted in FIG.6 for H, D and S. For example, if the side chains in the sequence haveless conformational freedom than the linear peptide, the entropy will bereduced. Such conformational restriction would enhance theconformational selectivity of antibodies specifically raised to thisantigen. The term “more constrained conformation” as used herein meansthat the dihedral angle distribution (ensemble of allowable dihedralangles) of one or more dihedral angles is at least 10% more constrainedthan in the comparator conformation, as determined for example by theentropy of the amino acids, for example H, and/or D (e.g. a moreconstrained conformation has lower entropy). Specifically, the averageentropy change relative to the entropy in the linear peptide,S(cyclic)-S(linear), of HDS in the overall more constrained cyclicconformational ensemble is on average reduced by more than 10% orreduced by more than 20% or reduced by more than 30% or reduced by morethan 40%, from the unconstrained conformational ensemble, e.g. of thequantity S(linear)-S(fibril)/[mean(S(linear)+S(fibril))] for the linearpeptide is approximately 81.7% entropy reduction for H6, 49.8% entropyreduction for D7, and −8.83% entropy reduction for S8 (the negativenumber implying that S8 has larger entropy for the cyclic peptide thanthe linear peptide).

The term “no or negligible plaque binding” or “lacks or has negligibleplaque binding” as used herein with respect to an antibody means thatthe antibody does not show typical plaque morphology staining onimmunohistochemistry (e.g. in situ) and the level of staining iscomparable to or no more than 2 fold the level seen with an IgG negative(e.g. irrelevant) isotype control

The term “Isolated peptide” refers to peptide that has been produced,for example, by recombinant or synthetic techniques, and removed fromthe source that produced the peptide, such as recombinant cells orresidual peptide synthesis reactants. The isolated peptide is optionally“purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purityand optionally pharmaceutical grade purity.

The term “detectable label” as used herein refers to moieties such aspeptide sequences (such a myc tag, HA-tag, V5-tag or NE-tag),fluorescent proteins that can be appended or introduced into a peptideor compound described herein and which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the label maybe radio-opaque, positron-emitting radionuclide (for example for use inPET imaging), or a radioisotope, such as ³H, ¹³N, ¹⁴C, ¹⁸F, ³²P, ³⁵S,¹²³I, ¹²⁵, ¹³¹I, a fluorescent (fluorophore) or chemiluminescent(chromophore) compound, such as fluorescein isothiocyanate, rhodamine orluciferin; an enzyme, such as alkaline phosphatase, beta-galactosidaseor horseradish peroxidase; an imaging agent; or a metal ion. Thedetectable label may be also detectable indirectly for example usingsecondary antibody.

The term “epitope” as commonly used means an antibody binding site,typically a polypeptide segment, in an antigen that is specificallyrecognized by the antibody. As used herein “epitope” can also refer tothe amino acid sequences or part thereof identified on A-beta using thecollective coordinates method described. For example an antibodygenerated against an isolated peptide corresponding to a cyclic compoundcomprising the identified target region HDSG SEQ ID NO:1), recognizespart or all of said epitope sequence. An epitope is “accessible” in thecontext of the present specification when it is accessible to binding byan antibody.

The term “greater affinity” as used herein refers to a relative degreeof antibody binding where an antibody X binds to target Y more strongly(K_(on)) and/or with a smaller dissociation constant (K_(off)) than totarget Z, and in this context antibody X has a greater affinity fortarget Y than for Z. Likewise, the term “lesser affinity” herein refersto a degree of antibody binding where an antibody X binds to target Yless strongly and/or with a larger dissociation constant than to targetZ, and in this context antibody X has a lesser affinity for target Ythan for Z. The affinity of binding between an antibody and its targetantigen, can be expressed as K_(A) equal to 1/K_(D) where K_(D) is equalto k_(on)/k_(off). The k_(on) and k_(off) values can be measured usingsurface plasmon resonance technology, for example using a MolecularAffinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg,Germany). An antibody that is selective for a conformation presented ina cyclic compound optional a cyclic peptide for example has a greateraffinity for the cyclic compound (e.g. cyclic peptide) compared to acorresponding sequence in linear form (e.g. the sequence non-cyclized).

Also as used herein, the term “immunogenic” refers to substances thatelicit the production of antibodies, activate T-cells and other reactiveimmune cells directed against an antigenic portion of the immunogen.

The term “corresponding linear compound” with regard to a cycliccompound refers to a compound, optionally a linear peptide, comprisingor consisting of the same sequence or chemical moieties as the cycliccompound but in linear (i.e. non-cyclized) form for example havingproperties as would be present in solution of a linear peptide. Forexample, the corresponding linear compound can be the synthesizedpeptide that is not cyclized.

As used herein “specifically binds” in reference to an antibody meansthat the antibody recognizes an epitope sequence and binds to its targetantigen with a minimum affinity. For example a multivalent antibodybinds its target with a K_(D) of at least 1e-6, at least 1e-7, at least1e-8, at least 1e-9, or at least 1e-10. Affinities greater than at least1e-8 may be preferred. An antigen binding fragment such as Fab fragmentcomprising one variable domain, may bind its target with a 10 fold or100 fold less affinity than a multivalent interaction with anon-fragmented antibody.

The term “selectively binds” as used herein with respect to an antibodythat selectively binds a form of A-beta (e.g. fibril, monomer oroligomer) or a cyclic compound means that the antibody binds the formwith at least 2 fold, at least 3 fold, or at least 5 fold, at least 10fold, at least 100 fold, at least 250 fold, at least 500 fold or atleast 1000 fold or more greater affinity. Accordingly an antibody thatis more selective for a particular conformation (e.g. oligomer)preferentially binds the particular form of A-beta with at least 2 foldetc greater affinity compared to another form and/or a linear peptide.

The term “linker” as used herein means a chemical moiety that can becovalently linked to the peptide comprising HDSG (SEQ ID NO: 1) epitopepeptide, optionally linked to HDSG (SEQ ID NO: 1) peptide N- andC-termini to produce a cyclic compound. The linker can comprise a spacerand/or one or more functionalizable moieties. The linker via thefunctionalizable moieties can be linked to a carrier protein or animmunogen enhancing agent such as Keyhole Limpet Hemocyanin (KLH).

The term “spacer” as used herein means any preferably non-immunogenic orpoorly immunogenic chemical moiety that can be covalently-linkeddirectly or indirectly to a peptide N- and C-termini to produce a cycliccompound of longer length than the peptide itself, for example thespacer can be linked to the N- and C-termini of a peptide consisting ofHDSG (SEQ ID NO: 1) to produce a cyclic compound of longer backbonelength than the HDSG (SEQ ID NO: 1) sequence itself. That is, whencyclized, the peptide with a spacer (for example of 3 amino acidresidues) makes a larger closed circle than the peptide without aspacer. The spacer may include, but is not limited to, moieties such asG, A, or PEG repeats, e.g. GHDSG (SEQ ID NO:7) GHDSGG (SEQ ID NO:8),GGHDSGG (SEQ ID NO:9), GHDSGGG (SEQ ID NO:10), etc. The spacer maycomprise or be coupled to one or more functionalizing moieties, such asone or more cysteine (C) residues, which can be interspersed within thespacer or covalently linked to one or both ends of the spacer. Where afunctionalizable moiety such as a C residue is covalently linked to oneor more termini of the spacer, the spacer is indirectly covalentlylinked to the peptide. The spacer can also comprise the functionalizablemoiety in a spacer residue as in the case where a biotin molecule isintroduced into an amino acid residue.

The term “functionalizable moiety” as used herein refers to a chemicalentity with a “functional group” which as used herein refers to a groupof atoms or a single atom that will react with another group of atoms ora single atom (so called “complementary functional group”) to form achemical interaction between the two groups or atoms. In the case ofcysteine, the functional group can be —SH which can be reacted to form adisulfide bond. Accordingly the linker can for example be CCC. Thereaction with another group of atoms can be covalent or a strongnon-covalent bond, for example as in the case of biotin-streptavidinbonds, which can have Kd˜1e-14. A strong non-covalent bond as usedherein means an interaction with a Kd of at least 1e-9, at least 1e-10,at least 1e-11, at least 1e-12, at least 1e-13 or at least 1e-14.

Proteins and/or other agents may be functionalized (e.g. coupled) to thecyclic compound, either to aid in immunogenicity, or to act as a probein in vitro studies. For this purpose, any functionalizable moietycapable of reacting (e.g. making a covalent or non-covalent but strongbond) may be used. In one specific embodiment, the functionalizablemoiety is a cysteine residue which is reacted to form a disulfide bondwith an unpaired cysteine on a protein of interest, which can be, forexample, an immunogenicity enhancing agent such as Keyhole LimpetHemocyanin (KLH), or a carrier protein such as Bovine serum albumin(BSA) used for in vitro immunoblots or immunohistochemical assays.

The term “reacts with” as used herein generally means that there is aflow of electrons or a transfer of electrostatic charge resulting in theformation of a chemical interaction.

The term “animal” or “subject” as used herein includes all members ofthe animal kingdom including mammals, optionally including or excludinghumans.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. Examples of conservative amino acid substitution include:

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferrednon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

For antibodies, percentage sequence identities can be determined whenantibody sequences maximally aligned by IMGT or other (e.g. Kabatnumbering convention). After alignment, if a subject antibody region(e.g., the entire mature variable region of a heavy or light chain) isbeing compared with the same region of a reference antibody, thepercentage sequence identity between the subject and reference antibodyregions is the number of positions occupied by the same amino acid inboth the subject and reference antibody region divided by the totalnumber of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentapplication may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. The nucleic acid can be either double stranded or singlestranded, and represents the sense or antisense strand. Further, theterm “nucleic acid” includes the complementary nucleic acid sequences aswell as codon optimized or synonymous codon equivalents. The term“isolated nucleic acid sequences” as used herein refers to a nucleicacid substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences which naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

“Operatively linked” is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate regulatory sequences is dependenton the host cell chosen and may be readily accomplished by one ofordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The term “vector” as used herein comprises any intermediary vehicle fora nucleic acid molecule which enables said nucleic acid molecule, forexample, to be introduced into prokaryotic and/or eukaryotic cellsand/or integrated into a genome, and include plasmids, phagemids,bacteriophages or viral vectors such as retroviral based vectors, AdenoAssociated viral vectors and the like. The term “plasmid” as used hereingenerally refers to a construct of extrachromosomal genetic material,usually a circular DNA duplex, which can replicate independently ofchromosomal DNA.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.-16.6 (Log10[Na+])+0.41 (% (G+C)-600/I), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm-5° C. based on the aboveequation, followed by a wash of 0.2× SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3× SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with early stageAD can be treated to prevent progression can be treated with a compound,antibody, immunogen, nucleic acid or composition described herein toprevent progression.

The term “administered” as used herein means administration of atherapeutically effective dose of a compound or composition of thedisclosure to a cell or subject.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve a desiredresult. Effective amounts when administered to a subject may varyaccording to factors such as the disease state, age, sex, weight of thesubject. Dosage regime may be adjusted to provide the optimumtherapeutic response.

The term “pharmaceutically acceptable” means that the carrier, diluent,or excipient is compatible with the other components of the formulationand not substantially deleterious to the recipient thereof.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and “the”include plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

II. Epitopes and Binding Proteins

The inventors have identified an “epitope region” in A-beta HDSG (SEQ IDNO: 1) at amino acid residues 6 to 9 of A-beta. They have furtheridentified that the epitope region may be or comprise a conformationalepitope, and that HDSG (SEQ ID NO: 1) may be selectively accessible toantibody binding in oligomeric species of A-beta.

Without wishing to be bound by theory, fibrils may present interactionsites that have a propensity to catalyze oligomerization. This may onlyoccur when selective fibril surface not present in normal individuals isexposed and able to have aberrant interactions with A-beta monomers.Environmental challenges such as low pH, osmolytes present duringinflammation, or oxidative damage may induce disruption in fibrils thatcan lead to exposure of more weakly stable regions. There is interest,then, to predict these weakly-stable regions, and use such predictionsto rationally design antibodies that could target them. Regions likelyto be disrupted in the fibril may also be good candidates for exposedregions in oligomeric species.

Computer based systems and methods to predict contiguous protein regionsthat are prone to disorder are described in U.S. Patent Application Ser.No. 62/253,044, SYSTEMS AND METHODS FOR PREDICTING MISFOLDED PROTEINEPITOPES BY COLLECTIVE COORDINATE BIASING filed Nov. 9, 2015 which ishereby incorporated by reference in its entirety. As described in theExamples, the methods were applied to A-beta and identified an epitopethat as demonstrated herein is specifically or selectively moreaccessible in A-beta oligomers.

As described in the Examples, cyclic peptide cyclo(CGHDSGG) (SEQ IDNO:2) may capture one or more of the conformational differences of theHDSG (SEQ ID NO: 1) epitope in oligomers relative to the monomer and/orfibril species. For example, differences in solvent accessible surfacearea, curvature, RMSD structural alignment, and the dihedral angledistributions for several of the amino acids and dihedral angles in thecyclic 7-mer cyclo (CGHDSGG) (SEQ ID NO:2) were found to besubstantially different than either the monomer and/or fibril,suggesting that the cyclic peptide provides for a conformational epitopethat is distinct from the linear epitope. Antibodies raised using animmunogen comprising cyclo(CGHDSGG) (SEQ ID NO:2) selectively boundcyclo(CGHDSGG) (SEQ ID NO:2) over linear CGHDSGG (SEQ ID NO:2) andselectively bound synthetic and/or native oligomeric A-beta speciescompared to monomeric A-beta and A-beta fibril plaques. Furtherantibodies raised to cyclo(CGHDSGG) were able to inhibit in vitropropagation of A-beta aggregation.

II. HDSG (SEQ ID NO: 1) “Epitope” Compounds

Accordingly, the present disclosure identifies an epitope in A-betaconsisting of amino acids HDSG (SEQ ID NO: 1) or a part thereof, HDSG(SEQ ID NO: 1) corresponding to amino acids residues 6-9 on A-beta. Asdemonstrated in the Examples, epitopes HDSG (SEQ ID NO:1), HDSGY (SEQ IDNO:4) and RHDSG (SEQ ID NO:5) (included in the epitopes collectivelyreferred to herein as “HDSG and related epitopes”) were identified asregions prone to disorder in an A-beta fibril. The residues HDSG (SEQ IDNO: 1) emerged in two predictions using the collective coordinatesmethod, while the flanking residues of this epitope, R5 and Y10, eachoccurred in one prediction.

An aspect includes a compound comprising an isolated A-beta peptidecomprising or consisting of HDSG (SEQ ID NO:1), sequence of a relatedepitope and/or part of any of the foregoing.

In an embodiment, the A-beta peptide is selected from an amino acidsequence comprising or consisting of HDSG (SEQ ID NO:1), HDSGY (SEQ IDNO:4) or RHDSG (SEQ ID NO:5). In an embodiment the A-beta peptide has asequence of an A-beta peptide as set forth in any one of the epitopes inTable 12. In an embodiment, the compound comprises the sequence as setforth in any of SEQ ID NO: 2, SEQ ID NOs:2, 28 and 29.

In an embodiment, the compound is a cyclic compound, such as acyclopeptide. The terms cyclopeptide and cyclic peptide are usedinterchangeably herein.

In some embodiments, the A-beta peptide comprising HDSG (SEQ ID NO: 1)(or a part thereof) can include 1, 2 or 3 additional residues present inA-beta, N- and/or C-terminus of HDSG (SEQ ID NO: 1) (or the partthereof), for example the A-beta peptide can include 1 residueN-terminal and be RHDSG (SEQ ID NO:5). As shown for example in theA-beta sequence of SEQ ID NO: 3, the 3 amino acids N-terminal to HDSG(SEQ ID NO:1) in A-beta are EFR and the 3 amino acids C-terminal to HDSG(SEQ ID NO: 1) are YEV. In embodiments, where the compound comprisingthe A-beta peptide is cyclized, the A-beta peptide is or is a maximum of8, A-beta residues, 7 A-beta residues or 6 A-beta residues. In anembodiment, the A-beta peptide is or is a maximum of 5 A-beta residues.For example, where the A-beta peptide is 6 amino acids it may compriseor consist of the amino acid sequence RHDSGY (SEQ ID NO:13), HDSGYE (SEQID NO: 11), DSGYEV (SEQ ID NO: 15) or FRHDSG (SEQ ID NO: 16).

In an embodiment, the compound further includes a linker. The linkercomprises a spacer and/or one or more functionalizable moieties. Thelinker can for example comprise 1, 2, 3, 4, 5, 6, 7 or 8 amino acidsand/or equivalently functioning molecules such as polyethylene glycol(PEG) moieties, and/or a combination thereof. In an embodiment, thespacer amino acids are selected from non-immunogenic or poorlyimmunogenic amino acid residues such as G and A, for example the spacercan be GGG, GAG, G(PEG)G, PEG-PEG-GG and the like. One or morefunctionalizable moieties e.g. amino acids with a functional group maybe included for example for coupling the compound to an agent ordetectable label or a carrier such as BSA or an immunogenicity enhancingagent such as KLH.

In an embodiment the linker comprises GC-PEG, PEG-GC, GCG or PEG-C-PEG.

In an embodiment, the linker comprises 2, 3, 4, 5, 6, 7 or 8 aminoacids.

In embodiments wherein the A-beta peptide comprising HDSG (SEQ ID NO: 1)or a part thereof includes 1, 2 or 3 additional residues found in A-betathat are N- and/or C-terminal to HDSG (SEQ ID NO: 1) the linker iscovalently linked to the N- and/or C-termini of the A-beta residues(e.g. where the peptide is RHDSG (SEQ ID NO:5), the linker is covalentlylinked to R and G residues). Similarly, where the A-beta peptide is HDSG(SEQ ID NO:1), the linker is covalently linked to residues H and G andwhere the A-beta peptide is HDSGY(SEQ ID NO:4), the linker is covalentlylinked to residues H and Y.

Proteinaceous portions of compounds (or the compound wherein the linkeris also proteinaceous) may be prepared by chemical synthesis usingtechniques well known in the chemistry of proteins such as solid phasesynthesis or synthesis in homogenous solution.

As mentioned, the compound can be a cyclic compound. Reference to the“cyclic peptide” herein can refer to a fully proteinaceous compound(e.g. wherein the linker is for example 1, 2, 3, 4, 5, 6, 7 or 8 aminoacids). It is understood that properties described for the cyclicpeptide determined in the examples can be incorporated in othercompounds (e.g. other cyclic compounds) comprising non-amino acid linkermolecules. The terms “cyclopeptide” and “cyclic peptide” are usedinterchangeably herein.

An aspect therefore provides a cyclic compound comprising peptide HDSG(SEQ ID NO: 1) (or a part thereof such as DSG) and a linker, wherein thelinker is covalently coupled directly or indirectly to the peptidecomprising HDSG (SEQ ID NO: 1) (e.g. the H and the G residues when thepeptide consists of HDSG (SEQ ID NO: 1)). In the cyclic compound forexample, at least H, D and/or S is in an alternate conformation than H,D and/or S in a corresponding linear peptide, optionally in a moreconstrained conformation.

In an embodiment, the cyclic compound comprises an A-beta peptidecomprising HDSG (SEQ ID NO: 1) and up to 6 A-beta residues (e.g. 1 or 2amino acids N and/or C terminus to HDSG (SEQ ID NO: 1)) and a linker,wherein the linker is covalently coupled directly or indirectly to thepeptide N-terminus residue and the C-terminus residue of the A-betapeptide. In the cyclic compound for example at least D is in analternate conformation than D in a corresponding linear peptide, or atleast S is in an alternate conformation than S in a corresponding linearpeptide and optionally wherein at least H, or at least D, is in a moreconstrained conformation than the conformation occupied in thecorresponding linear peptide comprising.

The cyclic compound can be synthesized as a linear molecule with thelinker covalently attached to the N-terminus or C-terminus of thepeptide comprising the A-beta peptide, optionally HDSG (SEQ ID NO:1) orrelated epitope, prior to cyclization. Alternatively part of the linkeris covalently attached to the N-terminus and part is covalently attachedto the C-terminus prior to cyclization. In either case, the linearcompound is cyclized for example in a head to tail cyclization (e.g.amide bond cyclization).

In an embodiment the cyclic compound comprises an A-beta peptidecomprising or consisting of HDSG (SEQ ID NO:1) and a linker, wherein thelinker is coupled to the N- and C-termini of the peptide (e.g. the H andthe G residues when the peptide consists of HDSG (SEQ ID NO:1).). In anembodiment, at least H, D and/or S is in an alternate conformation inthe cyclic compound than occupied by H, D and/or S in a linear compound,(e.g. linear peptide) comprising HDSG (SEQ ID NO: 1).

The linear peptide comprising the A-beta sequence, can be comprised in alinear compound. The linear compound or the linear peptide comprisingHDSG (SEQ ID NO: 1) is in an embodiment, a corresponding linear peptide.In another embodiment, the linear peptide is any length of A-betapeptide comprising HDSG (SEQ ID NO: 1), including for example a linearpeptide comprising A-beta residues 1-35, or smaller portions thereofsuch as A-beta residues 1-20, 2-20, 3-20, 1-15, 3-15, 3-12 and the likeetc. The linear peptide can in some embodiments also be a full lengthA-beta peptide.

In an embodiment, at least H, D and/or S is in an alternate conformationin the cyclic compound than occupied by a residue, optionally by Hand/or D, in the monomer and/or fibril.

In an embodiment, at least D, S and/or H is in an alternate conformationin the cyclic compound than occupied by a residue, optionally by Dand/or S, in the monomer and/or fibril.

In an embodiment, the alternate conformation is a constrainedconformation.

In an embodiment, at least H, optionally alone or in combination with atleast D is in a more constrained conformation than the conformationoccupied in a linear peptide comprising HDSG (SEQ ID NO: 1).

In an embodiment, the conformation of H and/or H in combination with oneor more of D and/or S is comprised in the compound in an alternateconformation, optionally in a more constrained conformation.

As shown in FIG. 6, residues H and D are in a more constrainedconformation in the cyclic compound compared to the conformationalensemble present in the linear peptide. The FIG. shows that there isapproximately a 81.7% entropy reduction for H and approximately a 49.8%entropy reduction for D. In an embodiment, the cyclic compound has aconformation H and/or D that is at least 10%, at least 20%, at least25%, at least 30%, at least 35% or at least 40% more constrainedcompared to a corresponding linear compound, as quantified by thatresidue's reduction in entropy.

For example, the alternate conformation can include one or morediffering dihedral angles in residues H, and/or D, and/or S, andoptionally in D and/or S differing from the dihedral angles in thelinear peptide and/or peptide in the context of the fibril.

As shown in FIG. 4, the dihedral angle distribution of D7 issubstantially different in the cyclic peptide compared to the linearpeptide or residue in the context of the fibril. In an embodiment, thecyclic compound comprises a D comprising an O-C-Cα-Cβ (also referred toas O-C-CA-CB) dihedral angle that is at least 30 degrees, at least 40degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees,at least 80 degrees, at least 90 degrees, at least 100 degrees, at least110 degrees, at least 120 degrees, at least 130 degrees, or at least 140degrees different, than the corresponding dihedral angle in the contextof the linear peptide and/or fibril. For example, Table 1 indicates thatfor simulated linear peptides, cyclic peptides, and fibrils, thedifference in this dihedral angle is about 160 degrees between cyclicand linear, and about 195 degrees between cyclic and fibril. Accordinglyin an embodiment, the cyclic compound comprises a D comprising anO-C-Cα-Cβ (also referred to as O-C-CA-CB) dihedral angle that is atleast 140 degrees different, at least 150 degrees different, at least160 degrees different, at least 170 degrees different, than thecorresponding dihedral angle in the context of the fibril.

Table 1 also identifies differences in the dihedral angle distributionsfor other angles, including those for example in residues H, D and S.

Accordingly in an embodiment the cyclic compound comprises an A-betapeptide residue selected from H, D and S, wherein at least one dihedralangle is at least 30 degrees, at least 40 degrees, at least 50 degrees,at least 60 degrees, at least 70 degrees, at least 80 degrees, at least90 degrees, at least 100 degrees, at least 110 degrees, at least 120degrees, at least 130 degrees or at least 140 degrees different, thanthe corresponding dihedral angle in the context of the linear compound.

In an embodiment, the cyclic compound comprises a minimum averageside-chain/backbone dihedral angle difference between the cycliccompound and linear peptide. For example, it is demonstrated for thecentroid conformations listed in Table 3, that the averageside-chain/backbone dihedral angle difference between the cyclic andlinear peptide is as follows H: 28.5 degrees, D: 133 degrees, S: 129degrees, G: 13 degrees. The corresponding numbers between the cyclic andfibril are H: 51 degrees, D: 103 degrees, S: 114 degrees, G: 73 degrees.

Accordingly, in an embodiment, the cyclic compound comprises an averageside-chain difference compared to the linear peptide of at least: for H,at least 20 degrees; for D and/or S at least 30 degrees, at least 40degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees,at least 80 degrees, at least 90 degrees, at least 100 degrees, at least110 degrees or at least 120 degrees; and/or for G, at least 30 degrees,at least 40 degrees, at least 50 degrees or at least 60 degrees.

The angle difference can for example be positive or negative, (+) or(−).

The alternate conformation can comprise an alternate backboneorientation. For example, the backbone orientation that the cyclicepitope exposes for an antibody differs compared to linear or fibrilform.

FIG. 7 plots the phi and psi angles sampled in equilibrium simulations,for residues H6, D7, S8, and G9 in both linear and cyclic peptidesconsisting of sequence CGHGSGG, as well as HDSG (SEQ ID NO: 1) in thecontext of the equilibrated fibril structure using initial conditionfrom PDB 2M4J. From FIG. 7 it is seen that the distribution of backbonedihedral angles (Ramachandran phi/psi angles) in the cyclic peptide isdifferent from the distribution of Ramachandran angles sampled foreither the linear peptide, or for the A-beta peptide HDSG (SEQ ID NO: 1)in the context the fibril structure 2M4J, particularly for residues D7and S8. Table 2 lists differences for peak values of distributions ofbackbone phi/psi angles. Similarly Table 3 shows backbone phi/phi anglesand the differences for the centroid structures (plotted in FIG. 10).For example, for the centroid conformations of the largest linearcluster and largest fibril cluster, for which dihedral angles are listedin Table 3, the average backbone Ramachandran angle difference (Δϕ,Δψ)between the cyclic and linear peptide is given as follows for HDSG (SEQID NO: 1): (−3.0,−1.1) degrees, (72.6, 164.1) degrees: (67.8, 195.1)degrees, (−38.8, 13.4) degrees; the average backbone Ramachandran angledifference (Δϕ, Δψ) between the cyclic and fibril peptide is given asfollows: (−53.7,29.7) degrees, (4.7,−154.5) degrees, (86.5,129) degrees,(127,−163.8) degrees.

Accordingly, in an embodiment, the cyclic compound comprises an A-betapeptide with at least one residue wherein backbone phi/psi angles is atleast 30 degrees, at least 40 degrees, at least 50 degrees, at least 60degrees, at least 70 degrees, at least 80 degrees different, at least 90degrees, at least 100 degrees, at least 110 degrees, at least 120degrees, at least 130 degrees, at least 140 degrees, at least 150degrees, at least 160 degrees, at least 170 degrees or at least 180degrees compared to the corresponding linear peptide or in a fibril PDBstructure.

The alternate conformation can also include an increase in curvaturecentered around an amino acid or of the cyclic compound comprising HDSG(SEQ ID NO: 1) or a related epitope relative to a corresponding linearpeptide and/or A-beta fibril.

In an embodiment, the alternate conformation HDSG (SEQ ID NO: 1) has anincreased curvature relative to linear HDSG (SEQ ID NO: 1). As shown inthe Examples, the curvature of the backbone at the positions of D7 andS8 in the cyclic compound is increased relative to the curvature atthose positions in the linear peptide, or peptide in the context of thefibril (FIG. 2) as described in Example 2.

The values of the curvature were determined for H, D, S, G incyclo(CGHDSGG) (SEQ ID NO:2), linear CGHDSGG (SEQ ID NO:2), and HDSG(SEQ ID NO:1) in the context of the fibril and are described in Example2.

Accordingly, the compound comprises an A-beta peptide wherein thecurvature of the D and/or S in the alternate conformation is increasedby at least 0.1, 0.2, 0.3 or more radians compared to the correspondinglinear peptide, or D7 or S8 in the context of the fibril.

In an embodiment, the HD, DS, SG, HDS, DSG, and/or HDSG (SEQ ID NO: 1)are in an alternate conformation, for example as compared to what isoccupied by these residues in a non-oligomeric conformation, such as thelinear peptide and/or fibril.

Further the entropy of the side chains is reduced in the cyclic peptiderelative to the linear peptide, rendering the side chains in a morestructured conformation than the linear peptide.

As demonstrated herein, the curvature of the cyclic compound is for someamino acids different than that in the linear peptide or to the peptidein the context of the fibril (FIG. 2). For example the curvature of D7in the context of the cyclic compound CGHDSGG (SEQ ID NO: 2) compared tothe corresponding linear peptide is increased.

Accordingly in one embodiment, the curvature of D and/or S in the cycliccompound is increased by at least 10%, at least 20%, or at least 30%compared to a corresponding linear compound.

It is also demonstrated, that one or more of the dihedral angles inresidues H, and/or D, and/or S, tend to be significantly different fromthe dihedral angles in the linear peptide or peptide in the context ofthe fibril. For these amino acids, when the solvent accessible surfacearea (SASA) is weighted by the solubility, more emphasis is placed onresidue S8. The entropy of the side chains of H6 is reduced in thecyclic peptide relative to the linear peptide and even the fibril,implying the tendency to have a restricted pose for this residue.

Cyclic compounds which show similar changes are also encompassed.

The cyclic compound in some embodiments that comprises a peptidecomprising HDSG (SEQ ID NO: 1) or related epitope can include 1, 2, 3 ormore residues in A-beta directly upstream and/or downstream of HDSG (SEQID NO: 1) or the related epitope. In such cases the spacer is covalentlylinked to the N- and C-termini of the ends of the corresponding residuesof the A-beta sequence.

In some embodiments, the linker or spacer is indirectly coupled to theN- and C-terminus residues of the A-beta peptide.

In an embodiment, the cyclic compound is a compound shown in FIG. 11B.

Methods for making cyclized peptides are known in the art and includeSS-cyclization or amide cyclization (head-to-tail, or backbonecyclization). Methods are further described in Example 3. For example, apeptide with “C” residues at its N- and C-termini, e.g. CGHDSGGC (SEQ IDNO: 2), can be reacted by SS-cyclization to produce a cyclic peptide.

As described in Example 2, a cyclic compound of FIG. 11B was assessedfor its relatedness to the conformational epitope identified. The cycliccompound comprising HDSG (SEQ ID NO: 1) peptide for example can be usedto raise antibodies selective for one or more conformational features.

The epitope HDSG (SEQ ID NO: 1) and/or a part thereof, as describedherein may be a potential target in misfolded propagating strains ofA-beta involved in A-beta, and antibodies that recognize theconformational epitope may for example be useful in detecting suchpropagating strains.

Also provided in another aspect is an isolated peptide comprising anA-beta peptide sequence described herein, including linear peptides andcyclic peptides. Linear peptides can for example be used for selectingantibodies for lack of binding thereto. The isolated peptide cancomprise a linker sequence described herein. The linker can becovalently coupled to the N or C terminus or may be partially coupled tothe N terminus and partially coupled to the C terminus as in CGHDSGG(SEQ ID NO: 2) linear peptide. In the cyclic peptide, the linker iscoupled to the C-terminus and N-terminus directly or indirectly.

Another aspect includes an immunogen comprising a compound, optionally acyclic compound described herein. The immunogen may also comprise forexample HDS, DSG or HDSG (SEQ ID NO: 1) or additional A-beta sequence.The amino acids may be directly upstream and/or downstream (i.e.N-terminal and/or C-terminal) of HDS, DSG or HDSG (SEQ ID NO: 1) orrelated epitope sequence. Antibodies raised against such immunogens canbe selected for example for binding to a cyclopeptide comprising HDSG(SEQ ID NO:1) or a related epitope.

A immunogen is suitably prepared or formulated for administration to asubject, for example, the immunogen may be sterile, or purified.

In an embodiment, the immunogen is a cyclic peptide comprising HDSG or arelated epitope.

In an embodiment, the immunogen comprises immunogenicity enhancing agentsuch as Keyhole Limpet Hemocyanin (KLH) or a MAP antigen. Theimmunogenicity enhancing agent can be coupled to the compound eitherdirectly, such as through an amide bound, or indirectly through afunctionalizable moiety in the linker. When the linker is a single aminoacid residue (for example with the A-beta peptide in the cyclic compoundis 6 amino acid residues) the linker can be the functionalizable moiety(e.g. a cysteine residue).

The immunogen can be produced by conjugating the cyclic compoundcontaining the constrained epitope peptide to an immunogenicityenhancing agent such as Keyhole Limpet Hemocyanin (KLH) or a carriersuch bovine serum albumin (BSA) using for example the method describedin Lateef et al 2007, herein incorporated by reference. In anembodiment, the method described in Example 3 or 4 is used.

A further aspect is an isolated nucleic acid molecule encoding theproteinaceous portion of a compound or immunogen described herein.

In embodiment, the nucleic acid molecule encodes any one of the aminoacid sequences sent forth in SEQ ID NOS: 1-16.

In an embodiment, nucleic acid molecule encodes HDSG (SEQ ID NO: 1) or arelated epitope and optionally a linker described herein.

A further aspect is a vector comprising said nucleic acid. Suitablevectors are described elsewhere herein.

III. Antibodies, Cells and Nucleic Acids

The compounds and particularly the cyclic compounds described above canbe used to raise antibodies that specifically bind DS, HDS or HDSG (SEQID NO: 1) in A-beta (e.g. residues 6-7, 6-8 or 6-9) and/or whichrecognize specific conformations of DS, HDSV or HDSG (SEQ ID NO: 1) inspecies of A-beta, for example oligomeric species of A-beta. Similarlycyclic compounds comprising for example RHDSG (SEQ ID NO: 5), HDSGY (SEQID NO: 4), HDSG (SEQ ID NO: 1) and/or other related epitope sequencesdescribed herein can be used to raise antibodies that specifically bindHDSG (SEQ ID NO: 1) etc and/or specific conformational epitopes thereof.As demonstrated herein, antibodies were raised to cyclo(CGHDSGG) (SEQ IDNO: 2), which specifically and/or selectively bound cyclo(CGHDSGG) (SEQID NO: 2) over linear CGHDSGG (SEQ ID NO: 2).

Accordingly as aspect includes an antibody (including a binding fragmentthereof) that specifically binds to an A-beta peptide having of asequence HDSG (SEQ ID NO: 1) or a related epitope sequence, for exampleas set forth in any one of SEQ ID NOs: 1 to 16.

In an embodiment, the A-beta peptide is comprised in a cyclic peptideand the antibody is specific or selective for A-beta presented in thecyclic compound.

In an embodiment, the antibody specially and/or selectively binds theA-beta peptide of the cyclic compound, wherein the A-beta has a sequenceas set forth in any one of SEQ ID NOs: 1 to 16.

In an embodiment, the cyclic compound is a cyclic peptide. In anembodiment, A-beta peptide in the cyclic peptide is any one of SEQ IDNO: 1-16. In a further embodiment, the cyclic peptide has a sequence asset forth in SEQ ID NO: 2, 12, 28 or 29.

As described in the examples, antibodies having one or properties can beselected using assays described in the Examples.

In an embodiment, the antibody does not bind a linear peptide comprisingthe sequence HDSG (SEQ ID NO: 1), optionally wherein the sequence of thelinear peptide is a linear version of a cyclic sequence used to raisethe antibody, optionally as set forth in SEQ ID NO: 2, 12, 28 or 29.

In an embodiment, the antibody is selective for the A-beta peptide aspresented in the cyclic compound relative to a corresponding linearcompound comprising the A-beta peptide.

In an embodiment, the antibody specifically binds an epitope on A-beta,the epitope comprising or consisting HDSG (SEQ ID NO: 1) or a relatedepitope thereof.

In an embodiment, the epitope recognized specifically or selectively bythe antibody on A-beta is a conformational epitope.

In an embodiment, the antibody is isolated.

In an embodiment, the antibody is an exogenous antibody

As described in the Examples, H, D, and/or S in the cyclic compound maybe predominantly accessible or exposed in conformations of A-beta thatare distinct from a corresponding linear peptide, monomer and/or fibrilforms.

Accordingly a further aspect is an antibody which specifically binds anepitope on A-beta, wherein the epitope comprises or consists of at leastone amino acid residue predominantly involved in binding to theantibody, wherein the at least one amino acid is H, D or S embeddedwithin the sequence HDSG (SEQ ID NO:1). In an embodiment, the epitopecomprises or consists of at least two consecutive amino acid residuespredominantly involved in binding to the antibody, wherein the at leasttwo consecutive amino acids are HD or DS or SG embedded within HDSG (SEQID NO:1).

In another embodiment, the epitope consists of HDSG (SEQ ID NO:1) or arelated epitope.

In an embodiment, the antibody is a conformation selective antibody. Inan embodiment, the antibody selectively binds a cyclic compoundcomprising an epitope peptide sequence described herein compared to thecorresponding linear sequence. For example an antibody that binds aparticular epitope conformation can be referred to as a conformationspecific antibody. Such antibodies can be selected using the methodsdescribed herein. The conformation selective antibody can differentiallyrecognize a particular A-beta species or a group of related species(e.g. dimers, trimers, and other oligomeric species) and can have ahigher affinity for one species or group of species compared to another(e.g. to either the monomer or fibril species).

In an embodiment, the antibody does not specifically bind monomericA-beta. In an embodiment, the antibody does not specifically bind A-betasenile plaques, for example in situ in AD brain tissue.

In another embodiment, the antibody does not selectively bind monomericA-beta compared to native- or synthetic-oligomeric A-beta.

For example, the antibody may specifically bind a cyclic compoundcomprises a residue selected from H, D and S, wherein at least onedihedral angle is at least 30 degrees, at least 40 degrees, at least 50degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees,at least 90 degrees, at least 100 degrees, at least 110 degrees, atleast 120 degrees, at least 130 degrees or at least 140 degreesdifferent in the cyclic compound, than the corresponding dihedral anglein the context of the linear compound.

In an embodiment, the antibody selectively binds A-beta peptide in acyclic compound, the A-beta comprising HDSG (SEQ ID NO: 1) or a partthereof, relative to a linear peptide comprising HDSG (SEQ ID NO: 1),such as a corresponding sequence. For example, in an embodiment theantibody selectively binds HDSG (SEQ ID NO: 1) in a cyclic conformationand has at least 2 fold, at least 3 fold, at least 5 fold, at least 10fold at least 20 fold, at least 30 fold, at least 40 fold, at least 50fold, at least 100 fold, at least 500 fold, at least 1000 fold greaterselectivity (e.g. greater binding affinity) for HDSG (SEQ ID NO: 1) inthe cyclic conformation compared to HDSG (SEQ ID NO: 1) in a linearpeptide, for example as measured by ELISA, or optionally a methoddescribed herein.

In an embodiment, the cyclic compound is cyclo(CGHDSGG) (SEQ ID NO: 2)or the cyclic compound with sequence as set forth in SEQ ID NO: 12, 28or 29.

In an embodiment, the antibody selectively binds A-beta peptide in acyclic compound and/or oligomeric A-beta. In an embodiment, theselectivity is at least 2 fold, at least 3 fold, at least 5 fold, atleast 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 100 fold, at least 500 fold, at least 1000 foldmore selective for the A-beta peptide in the cyclic compound and/orA-beta oligomer over a species of A-beta selected from A-beta monomerand/or A-beta fibril and/or a compound comprising a corresponding linearpeptide.

In an embodiment, the antibody lacks A-beta fibril plaque (also referredto as senile plaque) staining. Absence of plaque staining can beassessed by comparing to a positive control such as A-beta-specificantibodies 6E10 and 4G8 (Biolegend, San Diego, Calif.), or 2C8 (EnzoLife Sciences Inc., Farmingdale, N.Y.) and an isotype control. Anantibody described herein lacks or has negligible A-beta fibril plaquestaining if the antibody does not show typical plaque morphologystaining and the level of staining is comparable to or no more than 2fold the level seen with an IgG negative isotype control. The scale canfor example set the level of staining with isotype control at 1 and with6E10 at 10. An antibody lacks A-beta fibril plaque staining if the levelof staining on such a scale is 2 or less. In embodiment, the antibodyshows minimal A-beta fibril plaque staining, for example on theforegoing scale, levels scored at less about or less than 3.

In an embodiment, the antibody is a monoclonal antibody.

To produce monoclonal antibodies, antibody producing cells (Blymphocytes) can be harvested from a subject immunized with an immunogendescribed herein, and fused with myeloma cells by standard somatic cellfusion procedures thus immortalizing these cells and yielding hybridomacells. Such techniques are well known in the art, (e.g. the hybridomatechnique originally developed by Kohler and Milstein (Nature256:495-497 (1975)) as well as other techniques such as the human B-cellhybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., Methods Enzymol, 121:140-67 (1986)), and screening of combinatorialantibody libraries (Huse et al., Science 246:1275 (1989)). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with the epitope sequences described herein andthe monoclonal antibodies can be isolated.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries (see for example Ward et al., Nature41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990).

In an embodiment, the antibody is a humanized antibody.

The humanization of antibodies from non-human species has been welldescribed in the literature. See for example EP-B1 0 239400 and Carter &Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997 incorporated byreference in their entirety herein). Humanized antibodies are alsoreadily obtained commercially (e.g. Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.).

Humanized forms of rodent antibodies are readily generated by CDRgrafting (Riechmann et al. Nature, 332:323-327, 1988). In this approachthe six CDR loops comprising the antigen binding site of the rodentmonoclonal antibody are linked to corresponding human framework regions.CDR grafting often yields antibodies with reduced affinity as the aminoacids of the framework regions may influence antigen recognition (Foote& Winter. J Mol Biol, 224: 487-499, 1992). To maintain the affinity ofthe antibody, it is often necessary to replace certain frameworkresidues by site directed mutagenesis or other recombinant techniquesand may be aided by computer modeling of the antigen binding site (Co etal. J Immunol, 152: 2968-2976, 1994).

Humanized forms of antibodies are optionally obtained by resurfacing(Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach onlythe surface residues of a rodent antibody are humanized.

Human antibodies specific to a particular antigen may be identified by aphage display strategy (Jespers et al. Bio/Technology, 12: 899-903,1994). In one approach, the heavy chain of a rodent antibody directedagainst a specific antigen is cloned and paired with a repertoire ofhuman light chains for display as Fab fragments on filamentous phage.The phage is selected by binding to antigen. The selected human lightchain is subsequently paired with a repertoire of human heavy chains fordisplay on phage, and the phage is again selected by binding to antigen.The result is a human antibody Fab fragment specific to a particularantigen. In another approach, libraries of phage are produced weremembers display different human antibody fragments (Fab or Fv) on theirouter surfaces (Dower et al., WO 91/17271 and McCafferty et al., WO92/01047). Phage displaying antibodies with a desired specificity areselected by affinity enrichment to a specific antigen. The human Fab orFv fragment identified from either approach may be recloned forexpression as a human antibody in mammalian cells.

Human antibodies are optionally obtained from transgenic animals (U.S.Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach theheavy chain joining region (JH) gene in a chimeric or germ-line mutantmouse is deleted. Human germ-line immunoglobulin gene array issubsequently transferred to such mutant mice. The resulting transgenicmouse is then capable of generating a full repertoire of humanantibodies upon antigen challenge.

Humanized antibodies are typically produced as antigen binding fragmentssuch as Fab, Fab′ F(ab′)2, Fd, Fv and single domain antibody fragments,or as single chain antibodies in which the heavy and light chains arelinked by a spacer. Also, the human or humanized antibodies may exist inmonomeric or polymeric form. The humanized antibody optionally comprisesone non-human chain and one humanized chain (i.e. one humanized heavy orlight chain).

Antibodies, including humanized or human antibodies, are selected fromany class of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; andany isotype, including: IgG1, IgG2, IgG3 and IgG4. A chimeric, humanizedor human antibody may include sequences from one or more than oneisotype or class.

Additionally, antibodies specific for the epitopes described herein arereadily isolated by screening antibody phage display libraries. Forexample, an antibody phage library is optionally screened by usingcyclic compounds comprising peptides corresponding to epitopes disclosedherein to identify antibody fragments specific for conformation specificantibodies. Antibody fragments identified are optionally used to producea variety of recombinant antibodies that are useful with differentembodiments described herein. Antibody phage display libraries arecommercially available, for example, through Xoma (Berkeley, Calif.)Methods for screening antibody phage libraries are well known in theart.

A further aspect is antibody and/or binding fragment thereof comprisinga light chain variable region and a heavy chain variable region, theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, the light chain variable regioncomprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3and with the amino acid sequences of said CDRs comprising the sequencesset forth below:

SEQ ID NO: 17 CDR-H1 GYTFTSYW SEQ ID NO: 18 CDR-H2 IDPSDSQTSEQ ID NO: 19 CDR-H3 SRGGY SEQ ID NO: 20 CDR-L1 QDINNY SEQ ID NO: 21CDR-L2 YTS SEQ ID NO: 22 CDR-L3 LQYDNLWT

In an embodiment, the antibody is a monoclonal antibody. In anembodiment, the antibody is a chimeric antibody such as a humanizedantibody comprising the CDR sequences as recited in Table 10.

Also provided in another embodiment, is an antibody comprising the CDRsin Table 10 and a light chain variable region and a heavy chain variableregion, optionally in the context of a single chain antibody.

In yet another aspect, the antibody comprises a heavy chain variableregion comprises: i) an amino acid sequence as set forth in SEQ ID NO:24; ii) an amino acid sequence with at least 50%, at least 60%, at least70%, at least 80%, at least 90% sequence identity to SEQ ID NO: 24,wherein the CDR sequences are as set forth in SEQ ID NO: 17, 18 and 19,or iii) a conservatively substituted amino acid sequence i). In anotheraspect the antibody comprises a light chain variable region comprisingi) an amino acid sequence as set forth in SEQ ID NO: 26, ii) an aminoacid sequence with at least 50%, at least 60%, at least 70%, at least80% or at least 90% sequence identity to SEQ ID NO: 26, wherein the CDRsequences are as set forth in SEQ ID NO: 20, 21 and 22, or iii) aconservatively substituted amino acid sequence of i). In anotherembodiment, the heavy chain variable region amino acid sequence isencoded by a nucleotide sequence as set out in SEQ ID NO: 23 or a codondegenerate optimized version thereof. In another embodiment, theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 25 or a codondegenerate or optimized version thereof. In an embodiment, the heavychain variable region comprises an amino acid sequence as set forth inSEQ ID NO: 24

Another aspect is an antibody that specifically binds a same epitope asthe antibody with CDR sequences as recited in Table 10.

Another aspect includes an antibody that competes for binding to humanA-beta with an antibody comprising the CDR sequences as recited in Table10.

Competition between antibodies can be determined for example using anassay in which an antibody under test is assessed for its ability toinhibit specific binding of a reference antibody to the common antigen.A test antibody competes with a reference antibody if an excess of atest antibody (e.g., at least a 2 fold, 5, fold, 10 fold or 20 fold)inhibits binding of the reference antibody by at least 50%, at least75%, at least 80%, at least 90% or at least 95% as measured in acompetitive binding assay.

A further aspect is an antibody conjugated to a therapeutic, detectablelabel or cytotoxic agent. In an embodiment, the detectable label is apositron-emitting radionuclide. A positron-emitting radionuclide can beused for example in PET imaging.

A further aspect relates to an antibody complex comprising an antibodydescribed herein and/or a binding fragment thereof and oligomericA-beta.

A further aspect is an isolated nucleic acid encoding an antibody orpart thereof described herein.

Nucleic acids encoding a heavy chain or a light chain are also provided,for example encoding a heavy chain comprising CDR-H1, CDR-H2 and/orCDR-H3 regions described herein or encoding a light chain comprisingCDR-L1, CDR-L2 and/or CDR-L3 regions described herein.

The present disclosure also provides variants of the nucleic acidsequences that encode for the antibody and/or binding fragment thereofdisclosed herein. For example, the variants include nucleotide sequencesthat hybridize to the nucleic acid sequences encoding the antibodyand/or binding fragment thereof disclosed herein under at leastmoderately stringent hybridization conditions or codon degenerate oroptimized sequences In another embodiment, the variant nucleic acidsequences have at least 50%, at least 60%, at least 70%, most preferablyat least 80%, even more preferably at least 90% and even most preferablyat least 95% sequence identity to nucleic acid sequences encoding SEQ IDNOs: 24 and 26.

In an embodiment, the nucleic acid is an isolated nucleic acid.

Another aspect is an expression cassette or vector comprising thenucleic acid herein disclosed. In an embodiment, the vector is anisolated vector.

The vector can be any vector, including vectors suitable for producingan antibody and/or binding fragment thereof or expressing a peptidesequence described herein.

The nucleic acid molecules may be incorporated in a known manner into anappropriate expression vector which ensures expression of the protein.Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g. replication defective retroviruses,adenoviruses and adeno-associated viruses). The vector should becompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule encoding the peptidescorresponding to epitopes or antibodies described herein.

In an embodiment, the vector is suitable for expressing for examplesingle chain antibodies by gene therapy. The vector can be adapted forspecific expression in neural tissue, for example using neural specificpromoters and the like. In an embodiment, the vector comprises an IRESand allows for expression of a light chain variable region and a heavychain variable region.

Such vectors can be used to deliver antibody in vivo.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes.

Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector.

In an embodiment, the regulatory sequences direct or increase expressionin neural tissue and/or cells.

In an embodiment, the vector is a viral vector.

The recombinant expression vectors may also contain a marker gene whichfacilitates the selection of host cells transformed, infected ortransfected with a vector for expressing an antibody or epitope peptidedescribed herein.

The recombinant expression vectors may also contain expression cassetteswhich encode a fusion moiety (i.e. a “fusion protein”) which providesincreased expression or stability of the recombinant peptide; increasedsolubility of the recombinant peptide; and aid in the purification ofthe target recombinant peptide by acting as a ligand in affinitypurification, including for example tags and labels described herein.Further, a proteolytic cleavage site may be added to the targetrecombinant protein to allow separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to therecombinant protein.

Systems for the transfer of genes for example into neurons and neuraltissue both in vitro and in vivo include vectors based on viruses, mostnotably Herpes Simplex Virus, Adenovirus, Adeno-associated virus (AAV)and retroviruses including lentiviruses. Alternative approaches for genedelivery include the use of naked, plasmid DNA as well as liposome-DNAcomplexes. Another approach is the use of AAV plasmids in which the DNAis polycation-condensed and lipid entrapped and introduced into thebrain by intracerebral gene delivery (Leone et al. US Application No.2002076394).

Accordingly, in another aspect, the compounds, immunogens, nucleicacids, vectors and antibodies described herein may be formulated invesicles such as liposomes, nanoparticles, and viral protein particles,for example for delivery of antibodies, compounds, immunogens andnucleic acids described herein. In particular synthetic polymervesicles, including polymersomes, can be used to administer antibodies.

Also provided in another aspect is a cell, optionally an isolated and/orrecombinant cell, expressing an antibody described herein or comprisinga vector herein disclosed.

The recombinant cell can be generated using any cell suitable forproducing a polypeptide, for example suitable for producing an antibodyand/or binding fragment thereof. For example to introduce a nucleic acid(e.g. a vector) into a cell, the cell may be transfected, transformed orinfected, depending upon the vector employed.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins described herein may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells.

In an embodiment, the cell is a eukaryotic cell selected from a yeast,plant, worm, insect, avian, fish, reptile and mammalian cell.

In another embodiment, the mammalian cell is a myeloma cell, a spleencell, or a hybridoma cell.

In an embodiment, the cell is a neural cell.

Yeast and fungi host cells suitable for expressing an antibody orpeptide include, but are not limited to Saccharomyces cerevisiae,Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces andvarious species of the genus Aspergillus. Examples of vectors forexpression in yeast S. cerivisiae include pYepSecl, pMFa, pJRY88, andpYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for thetransformation of yeast and fungi are well known to those of ordinaryskill in the art.

Mammalian cells that may be suitable include, among others: COS (e.g.,ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No.CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1cells. Suitable expression vectors for directing expression in mammaliancells generally include a promoter (e.g., derived from viral materialsuch as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), aswell as other transcriptional and translational control sequences.Examples of mammalian expression vectors include pCDM8 and pMT2PC.

A further aspect is a hybridoma producing an antibody specific for anepitope described herein.

IV. Compositions

A further aspect is a composition comprising a compound, immunogen,nucleic acid, vector or antibody described herein.

In an embodiment, the composition comprises a diluent.

Suitable diluents for nucleic acids include but are not limited towater, saline solutions and ethanol.

Suitable diluents for polypeptides, including antibodies or fragmentsthereof and/or cells include but are not limited to saline solutions, pHbuffered solutions and glycerol solutions or other solutions suitablefor freezing polypeptides and/or cells.

In an embodiment, the composition is a pharmaceutical compositioncomprising any of the peptides, immunogens, antibodies, nucleic acids orvectors disclosed herein, and optionally comprising a pharmaceuticallyacceptable carrier.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, optionally as a vaccine, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. The composition may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Pharmaceutical compositions may comprise a pharmaceutically acceptablecarrier. Suitable pharmaceutically acceptable carriers includeessentially chemically inert and nontoxic compositions that do notinterfere with the effectiveness of the biological activity of thepharmaceutical composition. Examples of suitable pharmaceutical carriersinclude, but are not limited to, water, saline solutions, glycerolsolutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammoniumchloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), andliposomes. Such compositions should contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for direct administration to the patient.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In an embodiment comprising a compound or immunogen described herein,the composition comprises an adjuvant.

Adjuvants that can be used for example, include intrinsic adjuvants(such as lipopolysaccharides) that normally are the components of killedor attenuated bacteria used as vaccines. Extrinsic adjuvants areimmunomodulators which are typically non-covalently linked to antigensand are formulated to enhance the host immune responses. Aluminumhydroxide, aluminum sulfate and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants. A widerange of extrinsic adjuvants can provoke potent immune responses toimmunogens. These include saponins such as Stimulons (QS21, Aquila,Worcester, Mass.) or particles generated therefrom such as ISCOMs and(immunostimulating complexes) and ISCOMATRIX, complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria and mineral oil, Freund's completeadjuvant, bacterial products such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

In an embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is aluminum phosphate. Oil in water emulsionsinclude squalene; peanut oil; MF59 (WO 90/14387); SAF (SyntexLaboratories, Palo Alto, Calif.); and Ribi™ (Ribi Immunochem, Hamilton,Mont.). Oil in water emulsions may be used with immunostimulating agentssuch as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MOP),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide(™)), or other bacterial cell wallcomponents.

The adjuvant may be administered with an immunogen as a singlecomposition. Alternatively, an adjuvant may be administered before,concurrent and/or after administration of the immunogen.

Commonly, adjuvants are used as a 0.05 to 1.0 percent solution inphosphate-buffered saline. Adjuvants enhance the immunogenicity of animmunogen but are not necessarily immunogenic themselves. Adjuvants mayact by retaining the immunogen locally near the site of administrationto produce a depot effect facilitating a slow, sustained release ofimmunogen to cells of the immune system. Adjuvants can also attractcells of the immune system to an immunogen depot and stimulate suchcells to elicit immune responses. As such, embodiments may encompasscompositions further comprising adjuvants.

Adjuvants for parenteral immunization include aluminum compounds (suchas aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate). The antigen can be precipitated with, or adsorbed onto, thealuminum compound according to standard protocols. Other adjuvants suchas RIBI (ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

Adjuvants for mucosal immunization include bacterial toxins (e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof). For example, a purifiedpreparation of native cholera toxin subunit B (CTB) can be of use.Fragments, homologs, derivatives, and fusion to any of these toxins arealso suitable, provided that they retain adjuvant activity. Preferably,a mutant having reduced toxicity is used. Suitable mutants have beendescribed (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant)). Additional LT mutants that can be used in the methods andcompositions include, for example Ser-63-Lys, Ala-69-Gly, Glu-110-Asp,and Glu-112-Asp mutants. Other adjuvants (such as a bacterialmonophosphoryl lipid A (MPLA) of various sources (e.g., E. coli,Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri,saponins, or polylactide glycolide (PLGA) microspheres) can also be usedin mucosal administration.

Other adjuvants include cytokines such as interleukins for example IL-1,IL-2 and IL-12, chemokines, for example CXCL10 and CCLS, macrophagestimulating factor, and/or tumor necrosis factor. Other adjuvants thatmay be used include CpG oligonucleotides (Davis. Curr Top MicrobiolImmunol., 247:171-183, 2000).

Oil in water emulsions include squalene; peanut oil; MF59 (WO 90/14387);SAF (Syntex Laboratories, Palo Alto, Calif.); and Ribi™ (RibiImmunochem, Hamilton, Mont.). Oil in water emulsions may be used withimmunostimulating agents such as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide(TM)), or other bacterial cell wallcomponents.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol(3b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (forexample, U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (forexample, WO 88/9336).

An adjuvant may be coupled to an immunogen for administration. Forexample, a lipid such as palmitic acid, may be coupled directly to oneor more peptides such that the change in conformation of the peptidescomprising the immunogen does not affect the nature of the immuneresponse to the immunogen.

In an embodiment, the composition comprises an antibody describedherein. In another embodiment, the composition comprises an antibodydescribed herein and a diluent. In an embodiment, the composition is asterile composition.

V. Kits

A further aspect relates to a kit comprising i) an antibody and/orbinding fragment thereof, ii) a nucleic acid, iii) peptide or immunogen,iv) composition or v) recombinant cell described herein, comprised in avial such as a sterile vial or other housing and optionally a referenceagent and/or instructions for use thereof.

In an embodiment, the kit further comprises one or more of a collectionvial, standard buffer and detection reagent.

VI. Methods

Included are methods for making and using the compounds, immunogens andantibodies described herein.

In particular, provided are methods of making an antibody specificand/or selective for a conformational epitope of HDSG (SEQ ID NO: 1) orrelated epitope comprising administering to a subject, optionally anon-human subject, a conformationally restricted compound comprising anepitope sequence described herein, optionally cyclic compound comprisingHDSG (SEQ ID NO: 1) or related epitope, and isolating antibody producingcells or antibodies that specifically or selectively bind the cycliccompound and optionally i) specifically or selectively bind syntheticand/or native oligomers and/or that have no or negligible senile plaquebinding in situ tissue samples or no or negligible binding to acorresponding linear peptide. The cyclic compound can for examplecomprise any of the “epitopes” described herein containing cycliccompounds described herein.

In an embodiment, the method is for making a monoclonal antibody usingfor example a method as described herein.

In an embodiment, the method is for making a humanized antibody usingfor example a method described herein.

In an embodiment, the antibody is produced using a cyclic compound,optionally a cyclic peptide, described herein.

Antibodies produced using a cyclic compound are selected as describedherein and in the Examples. In an embodiment, the method comprisesisolating antibodies that specifically or selectively bind cyclicpeptide over linear peptide, are specific for the epitope sequence,specifically bind oligomer and/or lack or negligibly bind plaque in situand/or corresponding linear peptide, optionally using a method describedherein.

A further aspect provides a method of detecting whether a biologicalsample comprises A-beta, the method comprising contacting the biologicalsample with an antibody described herein and detecting the presence ofany antibody complex. In an embodiment, the method is for detectingwhether a biological sample comprises A-beta wherein at least H, Dand/or S is in an alternate conformation than occupied by H, D and/or Sin a non-oligomeric conformation.

In an embodiment the method is for detecting whether the biologic samplecomprises oligomeric A-beta.

In an embodiment, the method comprises:

-   -   a. contacting the biologic sample with an antibody described        herein that is specific and/or selective for A-beta oligomer        herein under conditions permissive to produce an antibody:A-beta        oligomer complex; and    -   b. detecting the presence of any complex; wherein the presence        of detectable complex is indicative that the sample may contain        A-beta oligomer.

In an embodiment, the level of complex formed is compared to a testantibody such as a suitable Ig control or irrelevant antibody.

In an embodiment, the detection is quantitated and the amount of complexproduced is measured. The measurement can for example be relative to astandard.

In an embodiment, the measured amount is compared to a control.

In another embodiment, the method comprises:

(a) contacting a biological sample of said subject with an antibodydescribed herein, under conditions permissive to produce anantibody-antigen complex;

(b) measuring the amount of the antibody-antigen complex in the testsample; and

(c) comparing the amount of antibody-antigen complex in the test sampleto a control;

wherein detecting antibody-antigen complex in the biological sample ascompared to the control indicates that the sample comprises A-beta.

The control can be a sample control (e.g. from a subject without AD, orfrom a subject with a particular form of AD, mild, moderate oradvanced), or be a previous sample from the same subject for monitoringchanges in A-beta oligomer levels in the subject.

In an embodiment, an antibody described herein is used.

In an embodiment, the antibody specifically and/or selectivelyrecognizes a conformation of A-beta comprising a HDSG (SEQ ID NO: 1) orrelated conformational epitope, and detecting the antibody antigencomplex in the biological sample is indicative that sample comprisesA-beta oligomer.

In an embodiment, the sample is a biological sample. In an embodiment,the sample comprises brain tissue or an extract thereof and/or CSF. Inan embodiment, the sample comprises whole blood, plasma or serum. In anembodiment, the sample is obtained from a human subject. In anembodiment, the subject is suspected of, at a risk of or has AD.

A number of methods can be used to detect an A-beta: antibody complexand thereby determine if A-beta comprising a HDSG (SEQ ID NO: 1) orrelated conformational epitope and/or A-beta oligomers is present in thebiological sample using the antibodies described herein, includingimmunoassays such as flow cytometry, immunoblots, ELISA, andimmunoprecipitation followed by SDS-PAGE, and immunocytochemistry.

As described in the Examples surface plasmon resonance technology can beused to assess conformation specific binding. If the antibody islabelled or a detectably labelled secondary antibody specific for thecomplex antibody is used, the label can be detected. Commonly usedreagents include fluorescent emitting and HRP labelled antibodies. Inquantitative methods, the amount of signal produced can be measured bycomparison to a standard or control. The measurement can also berelative.

A further aspect includes a method of measuring a level of- or imagingA-beta in a subject or tissue, optionally where the A-beta to bemeasured or imaged is oligomeric A-beta. In an embodiment, the methodcomprises administering to a subject at risk or suspected of having orhaving AD, an antibody conjugated to a detectable label; and detectingthe label, optionally quantitatively detecting the label. The label inan embodiment is a positron emitting radionuclide which can for examplebe used in PET imaging.

A further aspect includes a method of inducing an immune response in asubject, comprising administering to the subject a compound describedherein, optionally a cyclic compound comprising HDSG (SEQ ID NO:1) or arelated epitope peptide sequence, an immunogen and/or compositioncomprising said compound or said immunogen; and optionally isolatingcells and/or antibodies that specifically or selectively bind the A-betapeptide in the compound or immunogen administered. In an embodiment, thecomposition is a pharmaceutical composition comprising the compound orimmunogen in admixture with a pharmaceutically acceptable, diluent orcarrier.

In an embodiment, the subject is a non-human subject such as a rodent.Antibody producing cells generated are used in an embodiment to producea hybridoma cell line.

In an embodiment, the immunogen administered comprises a compoundillustrated in FIG. 11B.

It is demonstrated herein that antibodies raised against cyclo(CGHDSGG),can specifically and/or selectively bind A-beta oligomers and lackA-beta plaque staining. Oligomeric A-beta species are believed to be thetoxic propagating species in AD. Further as shown in FIG. 19, antibodyraised using cyclo(CGHDSGG) (SEQ ID NO: 2) and specific for oligomers,inhibited A-beta aggregation and A-beta oligomer propagation.Accordingly, also provided are methods of inhibiting A-beta oligomerpropagation, the method comprising contacting a cell or tissueexpressing A-beta with or administering to a subject in need thereof aneffective amount of an A-beta oligomer specific or selective antibodydescribed herein to inhibit A-beta aggregation and/or oligomerpropagation. In vitro the assay can be monitored as described in Example10.

The antibodies may also be useful for treating AD and/or other A-betaamyloid related diseases. For example, variants of Lewy body dementiaand in inclusion body myositis (a muscle disease) exhibit similarplaques as AD in the brain and muscle respectively, and A-beta can alsoform in aggregates implicated in cerebral amyloid angiopathy. Moreover,“mixed” pathology in neurodegenerative diseases (including Parkinson'sdisease and frontotemporal dementia) is recognized in which features ofAD pathology can be observed without a frank AD clinical syndrome. Asmentioned, antibodies raised to cyclo(CGHDSGG) (SEQ ID NO: 2) bindoligomeric A-beta which is believed to be a toxigenic species of A-betain AD and inhibit formation of toxigenic A-beta oligomers.

Accordingly a further aspect is a method of treating AD and/or otherA-beta amyloid related diseases, the method comprising administering toa subject in need thereof i) an effective amount of an antibodydescribed herein, optionally an A-beta oligomer specific or selective ora pharmaceutical composition comprising said antibody; or 2)administering an isolated cyclic compound comprising HDSG (SEQ ID NO:1)or a related epitope sequence or immunogen or pharmaceutical compositioncomprising said cyclic compound, to a subject in need thereof.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein. In an embodiment, a subject with detectable A-betalevels (e.g. A-beta antibody complexes measured in vitro or measured byimaging) is treated with the antibody.

The antibody and immunogens can for example be comprised in apharmaceutical composition as described herein, and formulated forexample in vesicles for improving delivery.

One or more antibodies targeting HDSG (SEQ ID NO:1) and/or relatedantibodies can be administered in combination. In addition theantibodies disclosed herein can be administered with one or more othertreatments such as a beta-secretase inhibitor or a cholinesteraseinhibitor.

In an embodiment, the antibody is a conformation specific/selectiveantibody, optionally that specifically or selectively binds A-betaoligomer.

Also provided are uses of the compositions, antibodies, isolatedpeptides, immunogens and nucleic acids for treating AD.

The compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids, vectors etc. described herein can be administered forexample, by parenteral, intravenous, subcutaneous, intramuscular,intracranial, intraventricular, intrathecal, intraorbital, ophthalmic,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol ororal administration.

In certain embodiments, the pharmaceutical composition is administeredsystemically.

In other embodiments, the pharmaceutical composition is administereddirectly to the brain or other portion of the CNS. For example suchmethods include the use of an implantable catheter and a pump, whichwould serve to discharge a pre-determined dose through the catheter tothe infusion site. A person skilled in the art would further recognizethat the catheter may be implanted by surgical techniques that permitvisualization of the catheter so as to position the catheter adjacent tothe desired site of administration or infusion in the brain. Suchtechniques are described in Elsberry et al. U.S. Pat. No. 5,814,014“Techniques of Treating Neurodegenerative Disorders by Brain Infusion”,which is herein incorporated by reference. Also contemplated are methodssuch as those described in US patent application 20060129126 (Kaplittand During “Infusion device and method for infusing material into thebrain of a patient”. Devices for delivering drugs to the brain and otherparts of the CNS are commercially available (e.g. SynchroMed® ELInfusion System; Medtronic, Minneapolis, Minn.).

In another embodiment, the pharmaceutical composition is administered tothe brain using methods such as modifying the compounds to beadministered to allow receptor-mediated transport across the blood brainbarrier.

Other embodiments contemplate the co-administration of the compositions,compounds, antibodies, isolated peptides, immunogens and nucleic acidsdescribed herein with biologically active molecules known to facilitatethe transport across the blood brain barrier.

Also contemplated in certain embodiments, are methods for administeringthe compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids described herein across the blood brain barrier suchas those directed at transiently increasing the permeability of theblood brain barrier as described in U.S. Pat. No. 7,012,061 “Method forincreasing the permeability of the blood brain barrier”, hereinincorporated by reference.

A person skilled in the art will recognize the variety of suitablemethods for administering the compositions, compounds, antibodies,isolated peptides, immunogens and nucleic acids described hereindirectly to the brain or across the blood brain barrier and be able tomodify these methods in order to safely administer the productsdescribed herein.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

Collective Coordinates Predictions

A method for predicting misfolded epitopes is provided by a methodreferred to as “Collective Coordinates biasing” which is described inU.S. patent application Ser. No. 62/253,044, SYSTEMS AND METHODS FORPREDICTING MISFOLDED PROTEIN EPITOPES BY COLLECTIVE COORDINATE BIASINGfiled Nov. 9, 2015, and is incorporated herein by reference. Asdescribed therein, the method uses molecular-dynamics-based simulationswhich impose a global coordinate bias on a protein (orpeptide-aggregate) to force the protein (or peptide-aggregate) tomisfold and then predict the most likely unfolded regions of thepartially unstructured protein (or peptide aggregate). Biasingsimulations were performed and the solvent accessible surface area(SASA) corresponding to each residue index (compared to that of theinitial structure of the protein under consideration). SASA represents asurface area that is accessible to H2O. A positive change in SASA(compared to that of the initial structure of the protein underconsideration) may be considered to be indicative of unfolding in theregion of the associated residue index. The method was applied to threeA-beta strains, each with its own morphology: a three-fold symmetricstructure of Aβ-40 peptides (or monomers) (PDB entry 2M4J), a two-foldsymmetric structure of Aβ-40 monomers (PDB entry 2LMN), and asingle-chain, parallel in-register (e.g. a repeated beta sheet where theresidues from one chain interact with the same residues from theneighboring chains) structure of Aβ-42 monomers (PDB entry 2MXU).

Simulations were performed for each initial structure using thecollective coordinates method as described in U.S. Patent ApplicationSer. No. 62/253,044 and the CHARMM force-field parameters described in:K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim,E. Darian, O. Guvench, P. Lopes, I. Vorobyov, and A. D. Mackerell.Charmm general force field: A force field for drug-like moleculescompatible with the charmm all-atom additive biological force fields.Journal of Computational Chemistry, 31(4):671-690, 2010; and P.Bjelkmar, P. Larsson, M. A. Cuendet, B. Hess, and E. Lindahl.Implementation of the CHARMM force field in GROMACS: analysis of proteinstability effects from correlation maps, virtual interaction sites, andwater models. J. Chem. Theo. Comp., 6:459-466, 2010, both of which arehereby incorporated herein by reference, with TIP3P water.

Epitopes predicted using this method are described in Example 2.

Example 2

I. Collective Coordinates Predictions

The epitope HDSG (SEQ ID NO:1) emerges as a predicted epitope fromstrain 2M4J from the collective coordinates approach described inExample 1. In several other strains of fibril, this region remainsdisordered and so has no structure in the PDB entry. For example in2LMN, HDS is unstructured and so coordinates for these residues are notpresent in the PDB structure, in 2LMP HDS is unstructured in the PDB,and in 2MXU HDSG (SEQ ID NO: 1) is unstructured in the PDB. Thecorresponding FIG. showing the predicted epitope is in FIG. 1. Forfibril structure 2M4J, 2 sequences bracketing HDSG (SEQ ID NO: 1) fromthe left and right, RHDSG (SEQ ID NO: 5) and HDSGY (SEQ ID NO: 4), arepredicted; residues R5 and Y10 each emerge from one prediction, whileresidues HDSG (SEQ ID NO: 1) emerge from 2 predictions, and so aretreated as a putative consensus sequence between these two predictions.

II. Conformation Specific Epitopes

As mentioned herein and shown in FIG. 1, the HDSG (SEQ ID NO: 1) epitopeemerges as a prediction upon adding denaturing stress to the fibril PDBstructure 2M4J. HDSGY (SEQ ID NO: 4) and RHSDG (SEQ ID NO: 5) also arepredicted.

Aβ is a peptide of length 36-43 amino acids that results from thecleavage of amyloid precursor protein (APP) by gamma secretase. In ADpatients, it is present in as multiple conformation monomers, insolublefibrils, and in soluble oligomers. Aβ fibril is the main component ofthe senile plaques found in the brains of AD patients.

In monomer form, Aβ exists as an unstructured polypeptide chain. Infibril form, Aβ can aggregate into distinct morphologies, often referredto as strains. Several of these structures have been determined bysolid-state NMR—some fibril structures have been obtained from in vitrostudies, and others obtained by seeding fibrils using amyloid plaquestaken from AD patients.

The oligomer is suggested to be a toxic and propagative species of thepeptide.

A prerequisite for the generation of oligomer-specific antibodies is theidentification of targets on Aβ peptide that are not present on or areless favourable in either the monomer or fibril conformations. Theseoligomer-specific epitopes would not differ in primary sequence from thecorresponding segment in monomer or fibril, however they would beconformationally distinct in the context of the oligomer. That is, theywould present a distinct conformation in the oligomer that would not bepresent in the monomer or fibril.

The structure of the oligomer has not been determined to date, moreover,NMR evidence indicates that the oligomer exists not in a singlewell-defined structure, but in a conformationally-plastic, malleablestructural ensemble with limited regularity. Moreover, the concentrationof oligomer species is far below either that of the monomer or fibril(estimates vary but on the order of 1000-fold below or more), makingthis target elusive.

Antibodies directed either against contiguous strands of primarysequence (e.g., linear sequence), or against fibril structures, maysuffer from several problems limiting their efficacy. Antibodies raisedto linear peptide regions tend not to be selective for oligomer, andthus bind to monomer as well. Because the concentration of monomer issubstantially higher than that of oligomer, such antibody therapeuticsmay suffer from “target distraction”, primarily binding to monomer andpromoting clearance of functional Aβ, rather than selectively targetingand clearing oligomeric species. Antibodies raised to amyloid inclusionsbind primarily to fibril, and have resulted in amyloid related imagingabnormalities (ARIA), including signal changes thought to representvasogenic edema and/or microhemorrhages.

To develop antibodies selective for oligomeric forms of Aβ, a regionthat may be disrupted in the fibril was identified. Without wishing tobe bound to theory, it was hypothesized that disruptions in the contextof the fibril may be exposed on the surface of the oligomer. Onoligomers however, these sequence regions may be exposed inconformations distinct from either that of the monomer and/or that ofthe fibril. For example, being on the surface, they may be exposed inturn regions that have higher curvature, higher exposed surface area,and different dihedral angle distribution than the correspondingquantities exhibit in either the fibril or the monomer.

Cyclic compounds comprising HDSG (SEQ ID NO: 1) are described herein andshown in FIG. 11B. The cyclic compounds have been designed to satisfyone or more of the above criteria of higher curvature, higher exposedsurface area, and alternative dihedral angle distributions.

A potential benefit of identifying regions prone to disruption in thefibril is that it may identify regions involved in secondary nucleationprocesses where fibrils may act as a catalytic substrate to nucleateoligomers from monomers [3]. Regions of fibril with exposed side chainsmay be more likely to engage in aberrant interactions with nearbymonomer, facilitating the accretion of monomers; such accreted monomerswould then experience an environment of effectively increasedconcentration at or near the surface of the fibril, and thus be morelikely to form multimeric aggregates including oligomers. Aged ordamaged fibril with exposed regions of Aβ may enhance the production oftoxic oligomer, and antibodies directed against these disordered regionson the fibril could be effective in blocking such propagativemechanisms.

III. Curvature of the Cyclic Peptide

The curvature profile of the cyclic peptide CGHDSGG (SEQ ID NO:2)differentiates cyclic HDSG (SEQ ID NO:1) from either the linear peptideor the fibril. The curvature profiles are shown in FIG. 2. The histidineresidue at position 6 has a lower curvature than either the linearpeptide or the fibril. The linear peptide tends to bend the backbonecompared to the conformations explored by either the cyclic peptide orfibril. On the other hand, the aspartic acid residue D7 and the serineresidue S8 have a higher curvature in the cyclic peptide compared to thecurvature of those residues in either the linear peptide or the fibril.The glycine residue (G) in the cyclic peptide has somewhat highercurvature than that in the fibril but a somewhat lower curvature thanthat in the linear peptide. These results imply that an antibodydirected against the cyclic peptide could show selectivity for a speciespresenting a different conformational ensemble than that of either themonomer or fibril.

For the plots of curvature, and dihedral angle distributions discussedherein, the data are obtained from equilibrium simulations in explicitsolvent (TIP3P) using the Charmm27. The simulation time and number ofconfigurations for each ensemble are as follows. Cyclic peptideensemble: simulation time 10 ns, containing 1001 frames; linear peptideensemble: simulation time 10 ns, containing 1001 frames; 2M4J ensemble:680 ps, containing 69 frames.

Because the curvature of the cyclic epitope has a different profile thaneither the linear peptide or fibril, it is expected that thecorresponding stretch of amino acids on an oligomer containing theseresidues would have a backbone orientation that is distinct from that inthe fibril or monomer. However the degree of curvature would not beunphysical—values of curvature characterizing the cyclic peptide areobtained in several locations of the fibril.

Numerical values of the curvature for residues H,D,S and G are given inTable 4. Note that the curvature for both the linear and cyclic peptidesis generally larger for HDSG (SEQ ID NO: 1) than the curvature of thoseresidues in the fibril, though not significantly larger than thecurvature in the fibril overall. This is largely due to the fact thatHDSG (SEQ ID NO: 1) is in a relatively extended beta strand conformationin the fibril structure 2M4J, and suggests that antibodies raised toeither the cyclic or linear peptides may be conformationally selectiveagainst the fibril, i.e. with low affinity to the fibril.

IV. Dihedral Angle Distributions

Further computational support for the identification of anoligomer-selective epitope, is provided by both the side chain dihedralangle distributions, and the Ramachandran ϕ and ψ distributions for thebackbone dihedral angles in the cyclic peptide a proxy for an exposedepitope in the oligomer—are for many angles substantially different fromthe corresponding distributions in either the fibril or monomer.

The side-chain dihedral distributions were examined for residues H, D,and S. The distributions of the C-C_(α)-C_(β)-C_(γ) andN-C_(α)-C_(β)-C_(γ) dihedral angles for H6 are different for the cyclicpeptide than for either the monomer or fibril distributions (FIG. 3).The probability that the linear peptide occupies a dihedral within therange of almost all (90%) of the cyclic peptide dihedral angles is 36%for either of the two above dihedrals, while the probability that thepeptide in the context of the fibril occupies a dihedral within therange of almost all (90%) of the cyclic peptide dihedral angles is only13% for either of the two above dihedrals. The other dihedral angleshave corresponding probabilities of approximately 30% or more for eitherthe fibril or monomer ensembles. In the following descriptions and FIG.sCA, Ca, or C_(α) are alternatively used to describe the C-alpha atom,and similarly for CB, Cb, and C_(β), and so on.

The dihedral distributions are shown for D7 in FIG. 4. The probabilitythat the linear peptide occupies a dihedral within the range of almostall (90%) of the cyclic peptide dihedral angles is as follows for thedihedral angles of D7:

-   C-CA-CB-CG: 12%-   N-CA-CB-CG: 19%-   O-C-CA-CB: 15%

The probability that the peptide in the context of the fibril occupies adihedral within the range of almost all (90%) of the cyclic peptidedihedral angles is as follows for the dihedral angles of D7:

-   C-CA-CB-CG: 30%-   N-CA-CB-CG: 49%-   O-C-CA-CB: 6%

The dihedral distributions are shown for S8 in FIG. 5. One can see thatthe dihedral angle distributions are substantially different for thedihedral angles in the cyclic conformational ensemble then they are ineither the linear or fibril ensembles. The probability that the linearpeptide occupies a dihedral within the range of almost all (90%) of thecyclic peptide dihedral angles is as follows for the dihedral angles ofS8:

-   C-CA-CB-OG: 92% (this indicates substantial overlap, but the weights    of the peaks having overlap in FIG. 5 are substantially different,    as can be seen from FIG. 5, indicating that the preferred dihedral    angles may still be different.-   N-CA-CB-OG: 84% (a similar comment applies to this dihedral angle as    does for C-CA-CB-OG above)-   O-C-CA-CB: 19%

The probability that the peptide in the context of the fibril occupies adihedral within the range of almost all (90%) of the cyclic peptidedihedral angles is as follows for the dihedral angles of S8:

-   C-CA-CB-OG: 30%-   N-CA-CB-OG: 28%-   O-C-CA-CB: 25%

According to the above analysis of side chain dihedral angledistributions, 7D AND 8S are the residues showing the largestdiscrepancy from the linear peptide and fibril ensembles. 7D and/or 8Smay be key residues on the epitope conferring conformationalselectivity.

Based on the data shown in FIGS. 3-5, Table 1 lists the peak values ofthe dihedral angle distributions, for those dihedral angles whosedistributions that show significant differences between the cyclicpeptide and other species. Column 1 in Table 1 is the specific dihedralconsidered, column 2 is the peak value of the dihedral distribution forthat angle in the context of the linear peptide CGHDSGG (SEQ ID NO: 2),column 3 is the peak value of the dihedral distribution for that anglein the context of the cyclic peptide CGHDSGG (SEQ ID NO: 2), column 4 isthe difference of the peak values of the dihedral distributions for thelinear and cyclic peptides, and column 5 is the peak value of thedihedral distribution for the peptide HDSG (SEQ ID NO: 1) in the contextof the fibril structure 2M4J.

TABLE 1 Peak Values of the Dihedral Angle Distributions DifferenceDihedral angle linear cyclic (linear-cyclic) fibril 6H: O-C-CA-CB −57.5,97.5 107.5 −165, −10 97.5 6H: CA-CB-CG-CD2 −107.5, 97.5  −82.5  −25, 180−107.5 6H: CA-CB-CG-ND1   77.5, −72.5 102.5  −25, −175 77.5, −77.5 6H:N-CA-CB-CG −67.5, 67.5, 180 −62.5 −5, 130, −117.5 67.5, 180   6H:C-CA-CB-CG 62.5, −57.5, 180 −57.5 120, 0, −122.5 −57.5, 180   7D:C-CA-CB-CG −77.5, 72.5, 172.5 172.5 97.5, −100, 0 72.5, −77.5, 180 7D:N-CA-CB-CG 57.5, −62.5, −162.5 −62.5 120, 0, −100 −62.5, 52.5  7D:O-C-CA-CB 102.5 −97.5 −160 97.5 8S: N-CA-CB-OG −172.5 −62.5 −110 62.58S: C-CA-CB-OG 67.5 180, 62.5 −112.5, 5    −72.5, 67.5  8S: O-C-CA-CB82.5 −102.5 −175 117.5V. Entropy of the Side Chains

The side chain entropy of a residue may be approximately calculated from

${S/k_{B}} = {- {\sum\limits_{i}{\int{d\;\phi_{i}{p\left( \phi_{i} \right)}\ln\mspace{11mu}{{p\left( \phi_{i} \right)}.}}}}}$

Where the sum is over all dihedral angles in a particular residue's sidechain, and p(Φ_(i)) is the dihedral angle distribution, as analyzedabove.

A plot of the increase in residue entropy in the cyclic peptideensemble, over the entropy of the fibril, is shown in FIG. 6. Theentropy of H6 is reduced compared to the linear and fibril, indicating amore constrained pose for H6. Similarly, the entropy of S8 is onlymarginally greater than the fibril for either the linear or cyclicpeptide. The entropy of D7 is reduced relative to the monomer butincreased relative to the fibril.

Dissection of Entropy of Residue Side-Chain Moieties

The entropy of each dihedral angle was investigated in the respectiveside chains of H, D, and S. The entropy of the dihedral angles for H6,D7, and S8 are plotted in FIG. 6. The entropy for several dihedrals ofH, D and S is reduced relative to the fibril, indicating a restrictedpose for those angles in a conformation that tends to be distinct fromeither the fibril or linear monomer.

The cyclic peptide is generally more rigid than the linear peptide,particularly for H6. Moreover, residue H6 is more rigid in the cyclicpeptide than in the fibril conformations. This suggests there may be awell-defined antigenic profile particularly around H6. The profile doeshave overlap however with the linear and fibril ensembles: theprobability of these ensembles to be within the top 90% of the H6distribution is as follows: (36%, 36%, 36%, 35%, 33%) and (13%, 13%,30%, 34%, 65%) for C-CA-CB-CG, N-CA-CB-CG, CA-CB-CG-ND1, CB-CG-ND1-CE1,and O-C-CA-CB in the cyclic-linear ensembles and cyclic-fibril ensemblesrespectively Low side chain conformational entropy in the cyclic peptidesupports a well-defined conformational pose that could aid in conferringselectivity.

VI. Ramachandran Angles

The backbone orientation that the epitope exposes to an antibody differsdepending on whether the peptide is in the linear, cyclic, or fibrilform. This discrepancy can be quantified by plotting the Ramachandranangles phi and psi (or ϕ and Φ), along the backbone, for residuesH,D,S,G in both the linear and cyclic peptides. FIG. 7 plots the phi andpsi angles sampled in equilibrium simulations, for residues H6, D7, S8,and G9 in both linear and cyclic peptides consisting of sequence CGHDSGG(SEQ ID NO: 2), as well as HDSG (SEQ ID NO: 1) in the context of thefibril structure 2M4J. From FIG. 7 it can be seen that the distributionof backbone dihedral angles in the cyclic peptide is most different fromthe distribution of dihedral angles sampled for either the linearpeptide, or for the peptide HDSG (SEQ ID NO: 1) in the context thefibril structure 2M4J, for the BB dihedral angles of residues D7 and S8.

As a specific example, for residue D7, FIG. 7 shows a distribution ofRamachandran ϕ, ψ angles that for the cyclic peptide has peak values(most-likely values) at (ϕ, ψ)((−65.9°,−44.5°). For the linear peptide,these most likely values are (ϕ, ψ)=(−153°,165°), and (−66°,143°) (thereare two peaks) and for the fibril structure 2M4J, these most likelyvalues are (ϕ, ψ)=(−60°,143°) and (−150°,143°). The substantialdifference in the peak dihedral angle values implies that antibodiesselected for the cyclic epitope conformation will likely have loweraffinity for the linear and fibril epitopes.

The peak values (most likely values) of the Ramachandran backbone ϕ, ψdistributions for H6, D7, S8, and G9 are given in Table 2. The firstcolumn in Table 2 gives the residue considered, which manifests twoangles, phi and psi, indicated in parenthesis. The 2^(nd) columnindicates the peak values of the Ramachandran phi/psi angles for HDSG(SEQ ID NO: 1) in the context of the linear peptide CGHDSGG (SEQ ID NO:2), while the 3^(rd) column indicates the peak values of theRamachandran phi/psi angles for HDSG (SEQ ID NO: 1) in the context ofthe cyclic peptide CGHDSGG (SEQ ID NO: 2), and the last column indicatesthe peak values of the Ramachandran phi/psi angles for HDSG (SEQ IDNO: 1) in the context of the fibril structure 2M4J. See FIG. 7. Thebackbone Ramachandran angles are very similar between all 3 species forH6. For D7, there are a minority of points in the linear and fibrilensembles that overlap with the points in the cyclic ensemble. If anellipse that encloses 90% of the points of the cyclic ensemble isconsidered, only about 16% of the linear ensemble is inside thisellipse, and only about 10% of the fibril ensemble is inside this hull.For analogous measures for H6, the corresponding numbers are 27% for thelinear ensemble and 32% for the fibril ensemble. For S8, the smallestconvex hull enclosing 90% of the points of the cyclic ensemble contains37% and 15% of the linear and fibril ensembles respectively. For G9, the90% convex hull is best split into two convex hulls containing 90% ofthe points. The fraction of points contained in these hulls is 79% fromthe linear ensemble, and 11% from the fibril ensemble.

TABLE 2 Peak values of distributions of backbone phi/psi angles Peakvalues of distributions of backbone phi/psi angles linear cyclic fibrilH6, (−98.5, 0)    (−164.6, 157.9)  (−147.5, 150)  (phi, psi) (−77.2,−43.3) (−85, 150) (−162.8, 157.4)  D7: (−153, 165)  (−65.9, −44.5)(−60.5, 143.5) (phi, psi)   (−66, 143.4) (−149.5, 143.5)  S8: (phi, psi)(−66.2, 144)   (−70.5, −50)   (−156, 174)  (−158.3, 151.1)  (−156, 12) (−66.5, −48)   G9: (85.9, 7) (86.1, −7) (−114.3, −14.6)  (77.5, 0)  (phi, psi) (77.5, 158)  (−62.3, 158)  VII. Solubility and Antigenicity of the Predicted Epitope Sequence

The solubility of the residues of A-beta 42 according to the CamSolprediction scheme [4] is shown in the FIG. 8. Residues H6-G9 are denotedby vertical lines.

The more soluble a residue is, the more likely it is to be encounteredon the surface of the oligomer. A relative solubility factor σi forresidue i is introduced, as:

$\sigma_{i} = \frac{s_{i} - s_{ave}}{\delta\; s}$where si is the solubility of residue i, s_(ave) is the averagesolubility of the 42-residue A-beta peptide, and δs, as given above, isthe standard deviation of the solubility of the 42 residue A-betapeptide.

A positive solubility indicates residues are more likely to be moreexposed to solvent and accessible to antibodies; the mean solubilityover all residues 1 through 42 in A-beta42 on this scale is −0.39. Inthe absence of further structural information, the increased solubilityof this region implies that it is likely to be exposed to solvent ratherthan buried. Thus in an ensemble of candidate oligomers, this region maytend to be more exposed than average. The CamSol method [4] employs alinear combination of specific physicochemical properties of aminoacids, including hydrophobicity, electrostatic charge of a residue atneutral pH, α-helix propensity, and β-strand propensity, which issmoothed over a window of seven residues to account for the effect ofthe neighboring residues. Solubility scores are computed asdimensionless numbers (A.U. or AU), and the solubility profiles arerescaled so that a random polypeptide yields a profile with mean 0 andstandard deviation 1. Accordingly, amino acids with a solubility scoresmaller than −1 are regarded as poorly soluble and have a negativeimpact on the solubility of a protein, while scores larger than 1 denotehighly soluble regions, yielding a positive contribution to the overallsolubility.

FIG. 9 plots the solvent accessible surface area (SASA), the SASAweighted by the solubility factor for each residue, σ, SASA, and σ,SASA, minus the value in the fibril, i.e. the increase in this quantityin the monomer and cyclic peptide over the fibril, σ_(i) ΔSASA_(i). Heres, is the solubility of residue i taken from FIG. 8, <s> is the averagesolubility over all 42 residues of A-beta, <s>=−0.39, and

${\delta\; s} = \sqrt{\frac{1}{N}{\sum\limits_{i = 1}^{42}\left( {{s_{i} -} < s >} \right)^{2}}}$is the standard deviation of the solubility across all 42 residues ofAβ. The plot of SASA vs residue index indicates that residues towardsthe N-terminus tend to display more antibody-accessible surface in allconformations. When weighted by the solubility to indicate a measure ofthe likelihood that a given residue would expose surface to solvent in acontext that has not been explicitly determined, residues H6 and S8 arecomparable in the cyclic peptide ensemble. When SASA is weighted by thesolubility as above, and then subtracted by the corresponding fibrilvalues to indicate values relative to the fibril, residue S8 emerges asmost exposed and soluble in the cyclic peptide. This analysis placesemphasis on residues S8 and H6 in this peptide as potentiallyparticularly important for binding.

The SASA of the cyclic and linear peptides are comparable, and bothlarger than the SASA in the fibril.

Weighting by the solubility results in the residue S8 having the mostlikelihood of differential exposure and availability for antibodybinding, as compared to the conformation of HDSG (SEQ ID NO: 1) in thefibril structure.

VIII. The Ensemble of Cyclic Peptide Conformations Clusters Differentlythan the Ensemble of Either Linear or Fibril Conformations

Definitive evidence that the sequence HDSG (SEQ ID NO: 1) displays adifferent conformation in the context of the cyclic peptide than in thelinear peptide can be seen by using standard structural alignmentmetrics between conformations, and then implementing clusteringanalysis. Equilibrium ensembles of conformations are obtained for thelinear and cyclic peptides CGHDSGG (SEQ ID NO: 2), as well as thefull-length fibril in the 3-fold symmetric structure corresponding toPDB ID 2M4J. Snapshots of conformations from these ensembles forresidues HDSG (SEQ ID NO: 1) are collected and then structurally alignedto the centroids of 3 largest clusters of the linear peptide ensemble,and the root mean squared deviation (RMSD) recorded. The clustering isperformed here by the maxcluster algorithm(http://www.sbg.bio.ic.ac.uk/maxcluster). The 3 corresponding RMSDvalues for the linear, cyclic, and fibril ensembles are plotted as a3-dimensional scatter plot in FIG. 10.

The cyclic peptide ensemble, shown as dark circles, is conformationallydistinct from either the linear peptide shown as grey crosses or fibrilensembles shown as grey inverted triangles. The top plot of FIG. 10shows that the cyclic peptide, but not the linear peptide, isdifferentiated from the conformations presented by the fibril. Thisimplies that an antibody raised to the cyclic peptide may beconformationally selective and may not preferentially bind the fibril orthe monomer, but that an antibody raised to the monomer may still bindto the fibril. Thus, without wishing to bound by theory, it may be thatif the cyclic peptide is used as a mimic of the oligomer, an antibodyraised to that mimic is unlikely to bind monomer or fibril.

It is evident from FIG. 10 that the 3 ensembles cluster differently fromeach other. In particular the cyclic peptide structural ensemble isdistinct from either the linear or fibril ensembles, implying thatantibodies specific to the cyclic peptide epitope will likely have lowaffinity to the conformations presented in the linear or fibrilensembles.

Two views of a representative snapshot, constituting the centroid of thelargest cluster from the cyclic peptide ensemble of structures, areshown in FIG. 11A. As well, the side-chain orientations that are presentfor a representative conformation in the linear peptide ensemble, havingdihedral angles near the peak of the dihedral angle distribution for thelinear peptide ensemble, are shown in black in FIG. 11A, superimposed onthe cyclic peptide, to make explicit their different orientations. Basedon the dihedral angle differences discussed above it is likely thatresidue D7, and to a somewhat lesser extent residue S8, will bedifferentially exposed.

FIG. 12 is a series of clustering plots by root mean squared deviation(RMSD) and the axes correspond to the centroids of the three largestclusters of the linear peptide ensemble, as in FIG. 10.

Each point corresponds to a given conformation taken from either thecyclic peptide, or various “strains” of fibril equilibrium ensembles,from PDB IDs 2LMN, 2MXU, and 2LMP. The cyclic peptide ensemble, shown asdark circles, is conformationally distinct from all the fibrilensembles. These fibrils all have varying degrees of disorderedN-termini, so that the fibril ensembles recapitulate to some extent thelinear ensemble. This implies that an antibody raised to the cyclicpeptide may be conformationally selective to not bind the fibrilconformations for multiple strains of A-beta

Table 3 lists values of the Ramachandran backbone and side chaindihedral angles undertaken for the cluster centroid cyclic peptideconformation taken from FIG. 10, and for the corresponding centroidconformations from the linear peptide and fibril ensembles. The centroidconformation for the largest cluster in the equilibrium fibril ensembleis also taken here. The differences of the corresponding dihedral anglesbetween the cyclic and linear conformations, and between the cyclic andfibril conformations are also given. The large majority of dihedralangles in this table are significantly different, as described herein.

TABLE 3 Table of Ramachandran backbone and side chain dihedral anglesshown for the cyclic peptide conformation that is the centroid of thelargest conformational cluster plotted in FIG. 10, and for the centroidsof the largest conformational clusters of the linear and fibrilensembles that are also plotted in FIG. 10. Cyclic linear 2m4jcyclic-linear cyclic-2m4j Rama-6H (−137.5, 154.4)  (−134.5, 155.5)(−83.8, 124.7) (−3.0, −1.1)  (−53.7, 29.7) Rama-7D (−75.2, −26.4)(−147.8, 137.7) (−79.9, 128.1) (72.6, 164.1)    (4.7, −154.5) Rama-8S(−73.5, −50.8) (−141.3, 144.3) (−160.0, −179.8) (67.8, 195.1)  (86.5,129) Rama-9G (−123.6, 10.3)  (−84.8, −3.1) (109.4, 174.1) (−38.8, 13.4)    (127, −163.8) 6H:O-C-CA-CB 103.2 100.4 65.9 2.8 37.3 6H:C-CA-CB-−68.9 48.9 57.2 −117.8 −126.1 CG 6H: N-CA-CB- 54.9 178.3 −177.8 −123.4−127.3 CG 6H: CA-CB-CG- 93.4 58.2 65.8 35.2 27.6 ND1 6H:CA-CB-CG- −100.3−127.6 −108.3 27.3 8 CD2 6H:CD2-CG- 170.3 −0.1 −6.23 170.4 176.53ND1-CE1 6H: CB-CG- 1.62 175 178.8 −173.38 −177.18 ND1-CE1 6H: NE2-CE1-−0.45 0.2 8.22 −0.65 −8.67 ND1-CG 6H: NE2-CD2- −2.12 0 2.58 −2.12 −4.7CG-ND1 6H: NE2-CD2- −169.6 −174.5 177.2 4.9 13.2 CG-CB 6H: ND1-CE1-−0.85 −0.25 −7.06 −0.6 6.21 NE2-CD2 6H: CG-CD2- 1.8 0.16 2.36 1.64 −0.56NE2-CE1 7D:O-C-CA-CB −94.9 87.9 68.9 177.2 −163.8 7D: C-CA-CB- 176.3−51.2 −180 −132.5 −3.6 CG 7D: N-CA-CB- −63.3 63.3 −60.9 −126.6 −2.4 CG7D: CA-CB-CG- −33.8 113.3 149.6 −147.1 176.6 OD2 7D: CA-CB-CG- 131.6−70.8 −37.3 −157.6 168.9 OD1 8S: N-CA-CB- −46.7 −163.2 −164 116.5 117.3OG 8S: C-CA-CB- −170.5 76.4 69.2 95.1 120.3 OG 8S: O-C-CA-CB −109.7 84.1125.8 166.2 124.5

TABLE 4 Table of mean curvature values for each residue in the cyclic,linear, and 2M4J fibril ensembles. Curvature vs residue index is plottedin FIG. 2. Curvatures Linear cyclic 2M4J 6H 1.19 0.781 1.01 7D 0.99 1.411.03 8S 0.95 1.36 0.93 9G 1.40 1.31 0.86

Example 3

Cyclic Compound Construction Comprising a Conformationally ConstrainedEpitope

Peptides comprising HDSG (SEQ ID NO: 1) such as Cyclo(CGHDSGG) (SEQ IDNO: 2) can be cyclized head to tail.

A linear peptide comprising HDSG (SEQ ID NO: 1) and a linker, preferablycomprising 2, 3, or 4 amino acids and/or PEG units, can be synthesizedusing known methods such as Fmoc based solid phase peptide synthesisalone or in combination with other methods. PEG molecules can be coupledto amine groups at the N terminus for example using coupling chemistriesdescribed in Ham ley 2014 [6] and Roberts et al 2012 [7], eachincorporated herein by reference. The linear peptide compound may becyclized by covalently bonding 1) the amino terminus and the carboxyterminus of the peptide+linker to form a peptide bond (e.g. cyclizingthe backbone), 2) the amino or carboxy terminus with a side chain in thepeptide+linker or 3) two side chains in the peptide+linker.

The bonds in the cyclic compound may be all regular peptide bonds(homodetic cyclic peptide) or include other types of bonds such asester, ether, amide or disulfide linkages (heterodetic cyclic peptide).

Peptides may be cyclized by oxidation of thiol- or mercaptan-containingresidues at the N-terminus or C-terminus, or internal to the peptide,including for example cysteine and homocysteine. For example twocysteine residues flanking the peptide may be oxidized to form adisulphide bond. Oxidative reagents that may employed include, forexample, oxygen (air), dimethyl sulphoxide, oxidized glutathione,cystine, copper (II) chloride, potassium ferricyanide, thallium(III)trifluro acetate, or other oxidative reagents such as may be known tothose of skill in the art and used with such methods as are known tothose of skill in the art.

Methods and compositions related to cyclic peptide synthesis aredescribed in US Patent Publication 2009/0215172. US Patent publication2010/0240865, US Patent Publication 2010/0137559, and U.S. Pat. No.7,569,541 describe various methods for cyclization. Other examples aredescribed in PCT Publication WO01/92466, and Andreu et al., 1994.Methods in Molecular Biology 35:91-169.

More specifically, a cyclic peptide comprising the HDSG (SEQ ID NO: 1)epitope can be constructed by adding a linker comprising a spacer withcysteine residues flanking and/or inserted in the spacer. The peptidecan be structured into a cyclic conformation by creating a disulfidelinkage between the non-native cysteines residues added to the N- andC-termini of the peptide. It can also be synthesized into a cycliccompound by forming a peptide bond between the N- and C-termini aminoacids (e.g. head to tail cyclization).

Peptide synthesis is performed by CPC Scientific Inc. (Sunnyvale Calif.,USA) following standard manufacturing procedures.

For example Cyclo(CGHDSGC)(SEQ ID NO: 12) cyclic peptide comprising theconformational epitope HDSG (SEQ ID NO: 1) is constructed in aconstrained cyclic conformation using a disulfide linkage betweencysteine residues added to the N- and C-termini of a peptide comprisingHDSG (SEQ ID NO: 1). Two non-native cysteine residues were added toGHDSG (SEQ ID NO: 7) one at the C-terminus and one at the N-terminus.The two cysteines are oxidized under controlled conditions to form adisulfide bridge or reacted head to tail to produce a peptide bond.

As described above, the structure of the cyclic peptide was designed tomimic the conformation and orientation of the amino acid side changes ofHDSG (SEQ ID NO: 1) in A-beta oligomer.

(SEQ ID NO: 2) Cyclo(CGHDSGG)

Cyclo(CGHDSGG) (SEQ ID NO: 2) was synthesized using the following method(CPC Scientific Inc, Sunnyvale Calif.). The protected linear peptide wassynthesized by standard conventional Fmoc-based solid-phase peptidesynthesis on 2-chlorotrityl chloride resin, followed by cleavage fromthe resin with 30% HFIP/DCM. Protected linear peptide was cyclized tothe corresponding protected cyclic peptide by using EDC.HCl/HOBt/DIEA inDMF at low concentration. The protected cyclic peptide was deprotectedby TFA to give crude cyclic peptide and the crude peptide was purifiedby RP HPLC to give pure cyclic peptide after lyophilize.

Cyclo(CGHDSGG) (SEQ ID NO: 2) can be prepared by amide condensation ofthe linear peptide CGHDSGG (SEQ ID NO: 2).

Cyclo(C-PEG2-HDSGG) can be prepared by amide condensation of the linearcompound C-PEG2-HDSGG (SEQ ID NO: 28).

Linear(CGHDSGG) was prepared (CPC Scientific Inc, Sunnyvale Calif.). Theprotected linear peptide was synthesized by standard conventionalFmoc-based solid-phase peptide synthesis on Fmoc-Gly-Wang resin, thenthe protected peptide was cleaved by TFA to give crude peptide and thecrude peptide was purified by RP HPLC to give pure peptide afterlyophilize, and which was used to conjugate BSA.

Immunogen Construction

The cyclic compound Cyclo(CGHDSGG) (SEQ ID NO: 2) was synthesized asdescribed above and then conjugated to BSA and/or KLH (CPC ScientificInc, Sunnyvale Calif.). BSA or KLH was re-activated by SMCC in PBSbuffer, then a solution of the pure peptide in PBS buffer was added tothe conjugation mixture, the conjugation mixture was stirred at roomtemperature (RT) for 2 h. Then the conjugation mixture was lyophilizedafter dialysis to give the conjugation product.

Example 4

Antibody Generation and Selection

A conformational constrained compound optionally a cyclic compound suchas a cyclic peptide comprising HDSG (SEQ ID NO: 1) such ascyclo(CGHDSGG) (SEQ ID NO: 2) peptide is linked to Keyhole LimpetHemocyanin (KLH). The cyclopeptide is sent for mouse monoclonal antibodyproduction (ImmunoPrecise Antibodies LTD (Victoria BC, Canada),following protocols approved by the Canadian Council on Animal Care.Mouse sera are screened using either the conformational peptide used forproducing the antibodies or a related peptide e.g. cyclo(CGHDSGG) (SEQID NO: 2) peptide, linked to BSA.

Hybridomas were made using an immunogen comprising cyclo(CGHDSGG) (SEQID NO: 2) as further described in Example 6. Hybridoma supernatants werescreened by ELISA and SPR for preferential binding to cyclo(CGHDSGG)(SEQ ID NO: 2) peptide vs linear (unstructured) peptide as describedherein. Positive IgG-secreting clones are subjected to large-scaleproduction and further purification using Protein G.

Example 5

Assessing Binding or Lack Thereof to Plaques/Fibrils

Immunohistochemistry can be performed on fresh frozen human brainsections, or frozen human brain sections, post fixed in 10% formalin.Endogenous peroxidase activity can be quenched using 0.5% hydrogenperoxide in methanol for 20 min. Antigen retrieval can be achieved usingsodium citrate pH 6.0 and steam heating for 25 min followed by coolingat room temperature (RT) for 30 min. After stabilization in TBS for 5-7min, sections are treated by 70% formic acid for 15 min at RT, and thenwashed 3×15 min in TBS. In a humidified chamber, non-specific stainingis blocked by incubation with serum-free protein blocking reagent (DakoCanada Inc., Mississauga, ON, Canada) for 1 h.

For immunostaining, antibodies described herein, positive control 6E10(1 μg/ml) and isotype controls IgG1, 2a and 2b (1 μg/ml, Abcam) are usedas primary antibodies. Sections are incubated overnight at 4° C., andwashed 3×5 min in TBS-T. Anti-mouse IgG Horseradish Peroxidaseconjugated (1:1000, ECL) is applied to sections and incubated 45 min,then washed 3×5 min in TBS-T. DAB chromogen reagent (VectorLaboratories, Burlington ON, Canada) is applied and sections rinsed withdistilled water when the desired level of target to background stainingis achieved. Sections are counterstained with Mayer's haematoxylin,dehydrated and cover slips were applied. Slides are examined under alight microscope (Zeiss Axiovert 200M, Carl Zeiss Canada, Toronto ON,Canada) and representative images captured at 50, 200 and 400×magnification using a Leica DC300 digital camera and software (LeicaMicrosystems Canada Inc., Richmond Hill, ON).

Example 6

Methods and Materials

Immunogen

Cyclic and linear peptides were generated at CPC Scientific, Sunnyvale,Calif., USA. Peptides were conjugated to KLH (for immunizing) and BSA(for screening) using a trifluoroacetate counter ion protocol. Peptideswere desalted and checked by MS and HPLC and deemed 95% pure. Peptideswere shipped to IPA for use in production of monoclonal antibodies inmouse.

Antibodies

A number of hybridomas and monoclonal antibodies were generated tocyclo(CGHDSGG) (SEQ ID NO: 2) linked to Keyhole Limpet Hemocyanin (KLH).

Fifty day old female BALB/c mice (Charles River Laboratories, Quebec)were immunized. A series of subcutaneous aqueous injections containingantigen but no adjuvant were given over a period of 19 days. Mice wereimmunized with 100 μg of peptide per mouse per injection of a 0.5 mg/mLsolution in sterile saline of cyclic peptide-KLH. Mice were housed in aventilated rack system from Lab Products. All 4 mice were euthanized onDay 19 and lymphocytes were harvested for hybridoma cell linegeneration.

Fusion/Hybridoma Development

Lymphocytes were isolated and fused with murine SP2/0 myeloma cells inthe presence of poly-ethylene glycol (PEG 1500). Fused cells werecultured using HAT selection. This method uses a semi-solidmethylcellulose-based HAT selective medium to combine the hybridomaselection and cloning into one step. Single cell-derived hybridomas growto form monoclonal colonies on the semi-solid media. 10 days after thefusion event, resulting hybridoma clones were transferred to 96-welltissue culture plates and grown in HT containing medium until mid-loggrowth was reached (5 days).

Hybridoma Analysis (Screening)

Tissue culture supernatants from the hybridomas were tested by indirectELISA on screening antigen (cyclic peptide-BSA) (Primary Screening) andprobed for both IgG and IgM antibodies using a Goatanti-IgG/IgM(H&L)-HRP secondary and developed with TMB substrate.Clones >0.2 OD in this assay were taken to the next round of testing.Positive cultures were retested on screening antigen to confirmsecretion and on an irrelevant antigen (Human Transferrin) to eliminatenon-specific mAbs and rule out false positives. All clones of interestwere isotyped by antibody trapping ELISA to determine if they are IgG orIgM isotype. All clones of interest were also tested by indirect ELISAon other cyclic peptide-BSA conjugates as well as linear peptide-BSAconjugates to evaluate cross-reactivity.

Mouse hybridoma antibodies were screened by Indirect ELISA usingcyclo(CGHDSGG) (SEQ ID NO: 2) conjugated to BSA.

ELISA Antibody Screening

Briefly, the ELISA plates were coated with 0.1 μg/wellcyclo(CGHDSGG)—conjugated-BSA (SEQ ID NO: 2) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4C and blocked with 3% skim milk powderin PBS for 1 hour at room temperature. Primary Antibody: Hybridomasupernatant at 100 uL/well incubated for 1 hour at 37 C with shaking.Secondary Antibody 1:10,000 Goat anti-mouse IgG/IgM(H+L)-HRP at 100uL/well in PBS-Tween for 1 hour at 37 C with shaking. All washing stepswere performed for 30 mins with PBS-Tween. The substrate3,3′,5,5′-tetramethylbenzidine (TMB) was added at 50 uL/well, developedin the dark and stopped with equal volume 1M HCl.

Positive clones were selected for further testing. Positive clones ofmouse hybridomas were tested for reactivity to cyclo(CGHDSGG) (SEQ IDNO: 2) conjugated BSA and human transferrin (HT) by indirect ELISA.Plates were coated with 1) 0.1 ug/well cyclo(CGHDSGG)—conjugated-BSA(SEQ ID NO: 2) at 10 uL/well in carbonate coating buffer (pH 9.6) O/N at4C; or 2) 0.25 ug/well HT Antigen at 50 uL/well in dH2O O/N at 37 C.Primary Antibody: Hybridoma supernatant at 100 uL/well incubated for 1hour at 37 C with shaking. Secondary Antibody 1:10,000 Goat anti-mouseIgG/IgM(H+L)-HRP at 100 uL/well in PBS-Tween for 1 hour at 37 C withshaking. All washing steps were performed for 30 mins with PBS-Tween.The substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added at 50uL/well, developed in the dark and stopped with equal volume 1M HCl.

ELISA Cyclo vs Linear CGHDSGG (SEQ ID NO: 2) Compound Selectivity

ELISA plates were coated with 1) 0.1 ug/wellcyclo(CGHDSGG)-conjugated—BSA (SEQ ID NO:2) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4 C; 2)) 0.1 ug/well linearCGHDSGG-conjugated-BSA (SEQ ID NO:2) at 100 uL/well in carbonate coatingbuffer (pH 9.6) O/N at 4 C; or 3) 0.1 ug/well Negative-Peptide at 100uL/well in carbonate coating buffer (pH 9.6) O/N at 4 C. PrimaryAntibody: Hybridoma supernatant at 100 uL/well incubated for 1 hour at37 C with shaking. Secondary Antibody 1:10,000 Goat anti-mouseIgG/IgM(H+L)-HRP at 100 uL/well in PBS-Tween for 1 hour at 37 C withshaking. All washing steps were performed for 30 mins with PBS-Tween.The substrate TMB was added at 50 uL/well, developed in the dark andstopped with equal volume 1M HCl.

Isotyping

The hybridoma antibodies were isotyped using antibody trap experiments.Trap plates were coated with 1:10,000 Goat anti-mouse IgG/IgM(H&L)antibody at 100 uL/well carbonate coating buffer pH9.6 overnight at 4 C.No blocking step was used. Primary antibody (hybridoma supernatants) wasadded (100 ug/mL). Secondary Antibody 1:5,000 Goat anti-mouse IgGy-HRPor 1:10,000 Goat anti-mouse IgMp-HRP at 100 uL/well in PBS-Tween for 1hour at 37 C with shaking. All washing steps were performed for 30 minswith PBS-Tween. The substrate TMB was added at 50 uL/well, developed inthe dark and stopped with equal volume 1M HCl.

SPR Binding Assays—Primary and Secondary Screens

SPR Analysis of Antibody Binding to Abeta Monomers and Oligomers

A-beta Monomer and Oligomer Preparation

Recombinant A-beta40 and 42 peptides (California Peptide, Salt Lake CityUtah, USA) were dissolved in ice-cold hexafluoroisopropanol (HFIP). TheHFIP was removed by evaporation overnight and dried in a SpeedVaccentrifuge. To prepare monomers, the peptide film was reconstituted inDMSO to 5 mM, diluted further to 100 μM in dH2O and used immediately.Oligomers were prepared by diluting the 5 mM DMSO peptide solution inphenol red-free F12 medium (Life Technologies Inc., Burlington ON,Canada) to a final concentration of 100 μM and incubated for 24 hours to7 days at 4° C.

SPR Analysis

All SPR measurements were performed using a Molecular Affinity ScreeningSystem (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany), an analyticalbiosensor that employs high intensity laser light and high speed opticalscanning to monitor binding interactions in real time. The primaryscreening of tissue culture supernatants was performed using an SPRdirect binding assay, whereby BSA-conjugated peptides, A-Beta42 Monomerand A-beta42 Oligomer are covalently immobilized on individual flowcells of a High Amine Capacity (HAC) sensorchip (Sierra Sensors GmbH,Hamburg, Germany) and antibodies flowed over the surface. Protein Gpurified mAbs were analyzed in a secondary screen using an SPR indirect(capture) binding assay, whereby the antibodies were captured on aprotein A-derivatized sensorchip (XanTec Bioanalytics GmbH, Duesseldorf,Germany) and A-Beta40 Monomer, A-beta42 Oligomer, soluble brain extractsand cerebrospinal fluid flowed over the surface. The specificity of theantibodies was verified in an SPR direct binding assay by covalentlyimmobilizing A-Beta42 Monomer and A-beta42 Oligomer on individual flowcells of a HAC sensorchip and flowing purified mAbs.

SPR Analysis of Soluble Brain Extracts and CSF Samples

Soluble brain extract and CSF Preparation

Human brain tissues and CSFs were obtained from patients assessed at theUBC Alzheimer's and Related Disorders Clinic. Clinical diagnosis ofprobable AD is based on NINCDS-ADRDA criteria [5]. CSFs are collected inpolypropylene tubes, processed, aliquoted into 100 μL polypropylenevials, and stored at −80° C. within 1 hour after lumbar puncture.

Homogenization: Human brain tissue samples were weighed and subsequentlysubmersed in a volume of fresh, ice cold TBS (supplemented withEDTA-free protease inhibitor cocktail from Roche Diagnostics, Laval QC,Canada) such that the final concentration of brain tissue is 20% (w/v).Tissue is homogenized in this buffer using a mechanical probehomogenizer (3×30 sec pulses with 30 sec pauses in between, allperformed on ice). TBS homogenized samples are then subjected toultracentrifugation (70,000×g for 90 min). Supernatants are collected,aliquoted and stored at −80° C. The protein concentration of TBShomogenates is determined using a BCA protein assay (PierceBiotechnology Inc, Rockford Ill., USA).

SPR Analysis

Brain extracts from 4 AD patients and 4 age-matched controls, and CSFsamples from 9 AD patients and 9 age-matched controls were pooled andanalyzed. Purified mAbs were captured on separate flow cells of aprotein A-derivatized sensor chip and diluted samples injected over thesurfaces for 180 seconds, followed by 120 seconds of dissociation inbuffer and surface regeneration. Binding responses weredouble-referenced by subtraction of mouse control IgG reference surfacebinding and assay buffer, and the different groups of samples compared

Assessing Binding or Lack Thereof to A-beta Monomers

In the primary screen of tissue culture supernatants, A-beta42 monomersand A-beta42 oligomers were used in a direct binding assay. In thesecondary screen, A-beta40 monomers and A-beta42 oligomers, solublebrain extracts and CSF samples were used in an indirect (capture)binding assay.

Primary Screen

Tissue culture supernatants were screened for the presence of antibodybinding against their cognate cyclic peptide. Each sample was dilutedand injected in duplicate over the immobilized peptide and BSA referencesurfaces for 120 seconds, followed by injection of running buffer onlyfor a 300-second dissociation phase. After every analytical cycle, thesensor chip surfaces were regenerated. Sensorgrams weredouble-referenced by subtracting out binding from the BSA referencesurfaces and blank running buffer injections, and binding responsereport points collected in the dissociation phase.

Oligomer Binding Assay

Next synthetic A-beta 42 oligomers were generated and immobilized asabove, antibody binding responses analyzed. Antibody binding responsesto A-beta 42 oligomers were compared to binding responses to cyclic.

Verifying Binding to A-beta Oligomers.

To further verify and validate A-beta42 Oligomer binding, antibodieswere covalently immobilized, followed by the injection over the surfaceof commercially-prepared stable A-beta42 Oligomers (SynAging SAS,Vandceuvre-les-Nancy, France).

Results

ELISA testing found that the majority of hybridoma clones bound thecyclopeptide.

Next clones were tested by ELISA for their binding selectivity forcyclo- and linear-CGHDSGG (SEQ ID NO: 2) compounds. A number of clonespreferentially bound cyclo(CGHDSGG)-conjugated-BSA (SEQ ID NO: 2)compared to linear CGHDSGG-conjugated—BSA (SEQ ID NO: 2).

Isotyping revealed that the majority of clones were IgG including IgG1,IgG2a and IgG3 clones. Several IgM and IgA clones were also identified,but not pursued further.

A direct binding analysis using surface plasmon resonance was performedto screen for antibodies in tissue culture supernatants that bind to thecyclic peptide of SEQ ID NO: 2.

FIG. 14 plots the results of the direct binding assay and the ELISAresults and shows that there is a correlation between the direct bindingand ELISA results.

Clones were retested for their ability to bind cyclic peptide, linearpeptide, A-beta 1-42 monomer and A-beta 1-42 oligomers prepared asdescribed above. Binding assays were performed using SPR as describedabove (Direct binding assays). A number of clones were selected based onthe binding assays performed as shown in Table 5.

The selected clones were IgG mAb. Negative numbers in the primary screenare indicative of no binding (e.g. less than isotype control).

TABLE 5 303 Cyclic- Linear- Aβ 42 Aβ 42 Peptide (RU) Peptide (RU)Monomer (RU) Oligomer (RU) 1B4 136.2 −0.1 56.5 109 2B10 171.9 −6.5 −4.169.8 3C2 74.9 −2.9 1.9 116.2 3C5 790.4 795.2 7.8 59 5E10 1334.9 35.7 8.260.2 6F1 23.4 −8.7 −11.6 77.9 8B2 310.1 7.6 −2.9 49.4 8E7 386.1 −4.2−25.1 54.2 9E5 253.5 −3.9 −20.1 50.8 10B9 17 −1.5 −23.2 61.9 10B10 235.2−4.6 −40.8 45.8 10G2 397.6 −0.7 61 109.8 11F10 148.8 −1.5 8.9 66.9ELISA Prescreen

The ELISA prescreen of hybridoma supernatants identified clones whichshowed increased binding to the cyclic peptides compared to the linearpeptide. A proportion of the clones were reactive to KLH-epitope linkerpeptide. These were excluded from further investigation. The majority ofthe clones were determined to be of the IgG isotype using the isotypingprocedure described herein.

Direct Binding Measured by Surface Plasmon Resonance—Primary Screen

Using surface plasmon resonance the tissue culture supernatantscontaining antibody clones were tested for direct binding to cyclicpeptide, linear peptide, A-beta oligomer and A-beta monomer.

The results for the primary screen are shown in FIG. 13. Panel A showsbinding to cyclic peptide and to linear peptide. Panel B shows bindingto A-beta oligomer and A-beta monomer. A number of the clones haveelevated reactivity to the cyclic peptide and all clones have minimal orno reactivity to linear peptide. There is a general selectivity forA-beta oligomer binding. Monomer reactivity is around or below 0 formost epitopes and most clones.

For select clones comparative binding profile is shown in FIG. 15. Eachclone is assessed for direct binding using surface plasmon resonanceagainst specific epitope in the context of cyclic peptide (structured),linear peptide (unstructured), A-beta monomer, and A-beta oligomer. Aclone reactive preferentially to unstructured epitope (e.g. linearepitope) was chosen as control, as indicated by an asterisk.

Example 7

Secondary Screen

Immunohistochemistry

Immunohistochemistry was performed on frozen human brain sections, withno fixation or antigen retrieval. In a humidified chamber, non-specificstaining was blocked by incubation with serum-free protein blockingreagent (Dako Canada Inc., Mississauga, ON, Canada) for 1 h. Thefollowing primary antibodies were used for immunostaining: mousemonoclonal isotype controls IgG1, IgG2a, and IgG2b, and anti-amyloidβ6E10, all purchased from Biolegend, and selected purified clonesreactive to the cyclopeptide. All antibodies were used at 1 μg/mL.Sections were incubated at room temperature for 1 h, and washed 3×5 minin TBS-T. Anti-Mouse IgG Horseradish Peroxidase conjugated (1:1000, ECL)was applied to sections and incubated 45 min, then washed 3×5 min inTBS-T. DAB chromogen reagent (Vector Laboratories, Burlington ON,Canada) was applied and sections rinsed with distilled water when thedesired level of target to background staining was achieved. Sectionswere counterstained with Mayer's haematoxylin, dehydrated and coverslips were applied. Slides were examined under a light microscope (ZeissAxiovert 200M, Carl Zeiss Canada, Toronto ON, Canada) and representativeimages captured at 20 and 40× magnification using a Leica DC300 digitalcamera and software (Leica Microsystems Canada Inc., Richmond Hill, ON).Images were optimized in Adobe Photoshop using Levels Auto Correction.

CSF and Brain Extracts

Human brain tissues were obtained from the University of Maryland Brainand Tissue Bank upon approval from the UBC Clinical Research EthicsBoard (C04-0595). CSFs were obtained from patients assessed at the UBCHospital Clinic for Alzheimer's and Related Disorders. The study wasapproved by the UBC Clinical Research Ethics Board, and written consentfrom the participant or legal next of kin was obtained prior tocollection of CSF samples. Clinical diagnosis of probable AD was basedon NINCDS-ADRDA criteria. CSFs were collected in polypropylene tubes,processed, aliquoted into 100 μL polypropylene vials, and stored at −80°C. within 1 hour after lumbar puncture.

Homogenization: Human brain tissue samples were weighed and subsequentlysubmersed in a volume of fresh, ice cold TBS and EDTA-free proteaseinhibitor cocktail from Roche Diagnostics (Laval QC, Canada) such thatthe final concentration of brain tissue was 20% (w/v). Tissue washomogenized in this buffer using a mechanical probe homogenizer (3×30sec pulses with 30 sec pauses in between, all performed on ice). TBShomogenized samples were then subjected to ultracentrifugation (70,000×gfor 90 min). Supernatants were collected, aliquoted and stored at −80°C. The protein concentration of TBS homogenates was determined using aBCA protein assay (Pierce Biotechnology Inc, Rockford Ill., USA).

CSF: CSF was pooled from 9 donors with AD and 9 donors without AD.Samples were analyzed by SPR using purified IgG at a concentration of 30micrograms/ml for all antibodies Mouse IgG was used as an antibodycontrol, and all experiments were repeated at least 2 times.

Positive binding in CSF and brain extracts was confirmed using antibody6E10.

SPR Analysis: 4 brain extracts from AD patients and 4 brain extractsfrom age-matched controls were pooled and analyzed. Brain samples,homogenized in TBS, included frontal cortex Brodmann area 9. Allexperiments were performed using a Molecular Affinity Screening System(MASS-1) (Sierra Sensors GmbH, Hamburg, Germany), an analyticalbiosensor that employs high intensity laser light and high speed opticalscanning to monitor binding interactions in real time as described inExample 6. Purified antibodies generated for cyclopeptides describedherein were captured on separate flow cells of a protein A-derivatizedsensor chip and diluted samples injected over the surfaces for 180seconds, followed by 120 seconds of dissociation in buffer and surfaceregeneration. Binding responses were double-referenced by subtraction ofmouse control IgG reference surface binding and assay buffer, and thedifferent groups of samples compared.

Results

CSF Brain Extracts and Immunohistochemistry

Several clones were tested for their ability to bind A-beta in CSF,soluble brain extracts and tissue samples of cadaveric AD brains areshown in Table 6. Strength of positivity in Table 6 is shown by thenumber plus signs.

Table 6 and Table 7 provide data for selected clone's bindingselectivity for oligomers over monomer measured as described herein bySPR.

IHC results are also summarized in Table 6 where “+/−” denotes stainingsimilar to or distinct from isotype control but without clear plaquemorphology.

FIG. 16 shows an example of the lack of plaque staining on fresh frozensections with clone 25-1B4 compared to the positive plaque staining seenwith 6E10 antibody.

FIG. 17 shows, antibodies raised to the cyclopeptide comprising HDSG(SEQ ID NO: 1) bound A-beta oligomer preferentially over monomer andalso preferentially bound A-beta in brain extracts and/or CSF of ADpatients.

As shown in Tables 6, 7 and FIGS. 16 and 17, antibodies raised to thecyclopeptide comprising HDSG (SEQ ID NO: 1) bound to A-beta in brainextracts and/or CSF, but did not appreciably bind to monomers on SPR,and did not appreciably bind to plaque fibrils by IHC

TABLE 6 Summary of binding characteristics Oligomers/ CSF Brain ExtractIHC - Plaque Clone # Monomers AD/Non-AD AD/Non-AD Staining cyclo(CGHDSGG) 25 (1B4) +++ +++ + − (SEQ ID NO: 2) 28 (3C5) + − ++ + 26 ++ −++ − 30 + − ++ N/A * Scoring is relative to other clones in the samesample category.

TABLE 7 A-beta Oligomer binding RU values subtracted for monomer bindingClone tested 303-3C5 RU 227.7

Example 8

Synthetic Oligomer Binding

Serial 2-fold dilutions (7.8 nM to 2000 nM) of commercially-preparedsynthetic amyloid beta oligomers (SynAging SAS, Vandceuvre-lés-Nancy,were tested for binding to covalently immobilized antibodies. Resultsfor control antibody mAb6E10 is shown in FIG. 18A and for mouse controlIgG is shown in FIG. 18B. FIG. 18C shows results using an antibodyraised against cyclo(CGHDSGG) (SEQ ID NO:2).

Example 9

Immunohistochemistry on Formalin Fixed Tissues

Human brain tissue was assessed using antibodies raised tocyclo(CGHDSGG) (SEQ ID NO: 2). The patient had been previouslycharacterized and diagnosed with Alzheimer's disease with a tripartiteapproach: (i) Bielschowsky silver method to demonstrate senile plaquesand neurofibrillary tangles, (ii) Congo red to demonstrate amyloid and(iii) tau immunohistochemistry to demonstrate tangles and to confirm thesenile plaques are “neuritic”. This tissue was used to test plaquereactivity of selected monoclonal antibody clones. The brain tissueswere fixed in 10% buffered formalin for several days and paraffinprocessed in the Sakura VIP tissue processors. Tissue sections wereprobed with 1 μg/ml of antibody with and without microwave antigenretrieval (AR). The pan-amyloid beta reactive antibody 6E10 was includedalong with selected antibody clones as a positive control. Antibodieswere diluted in Antibody Diluent (Ventana), color was developed withOptiView DAB (Ventana). The staining was performed on the VentanaBenchmark XT IHC stainer. Images were obtained with an Olympus BX45microscope. Images were analyzed blind by a professional pathologistwith expertise in neuropathology.

As shown in Table 8 below, using fixed tissue, the tested antibodieswere negative for specific staining of senile plaque amyloid with orwithout antigen retrieval. 6E10 was used as the positive control.

TABLE 8 Convincing evidence of specific staining of senile plaqueamyloid Epitope Antibodies to test Without AR Plus AR 303 25 Neg Neg 28Neg Neg Positive Control 6E10 strongly strongly positive positive

Example 10

Inhibition of Oligomer Propagation

The biological functionality of antibodies was tested in vitro byexamining their effects on propagation of Amyloid Beta (Aβ) aggregationusing the Thioflavin T (ThT) binding assay. Aβ aggregation is induced byand propagated through nuclei of preformed small Aβ oligomers, and thecomplete process from monomeric Aβ to soluble oligomers to insolublefibrils is accompanied by concomitantly increasing beta sheet formation.This can be monitored by ThT, a benzothiazole salt, whose excitation andemission maxima shifts from 385 to 450 nm and from 445 to 482 nmrespectively when bound to beta sheet-rich structures and resulting inincreased fluorescence. Briefly, Aβ 1-42 (Bachem Americas Inc.,Torrance, Calif.) was solubilized, sonicated, diluted in Tris-EDTAbuffer (pH7.4) and added to wells of a black 96-well microtitre plate(Greiner Bio-One, Monroe, N.C.) to which equal volumes of cyclopeptideraised antibody or irrelevant mouse IgG antibody isotype controls wereadded, resulting in a 1:5 molar ratio of Aβ1-42 peptide to antibody. ThTwas added and plates incubated at room temperature for 24 hours, withThT fluorescence measurements (excitation at 440 nm, emission at 486 nm)recorded every hour using a Wallac Victor3v 1420 Multilabel Counter(PerkinElmer, Waltham, Mass.). Fluorescent readings from backgroundbuffer were subtracted from all wells, and readings from antibody onlywells were further subtracted from the corresponding wells.

As shown in FIG. 19, Aβ42 aggregation, as monitored by ThT fluorescence,demonstrated a sigmoidal shape characterized by an initial lag phasewith minimal fluorescence, an exponential phase with a rapid increase influorescence and finally a plateau phase during which the Aβ molecularspecies are at equilibrium and during which there is no increase influorescence. Co-incubation of Aβ42 with an irrelevant mouse antibodydid not have any significant effect on the aggregation process. Incontrast, co-incubation of Aβ42 with the test antibodies completelyinhibited all phases of the aggregation process. Results obtained withantibody clone 25 (1B4; IgG2a isotype) are shown in FIG. 19. As the ThTaggregation assay mimics the in vivo biophysical/biochemical stages ofAβ propagation and aggregation from monomers, oligomers, protofibrilsand fibrils that is pivotal in AD pathogenesis, the antibodies raised tocyclo CGHDSGG demonstrate the potential to completely abrogate thisprocess. Isotype control performed using IgG2a showed no inhibition.

Example 11

Achieving the Optimal Profile for Alzheimer's Immunotherapy: RationalGeneration of Antibodies Specific for Toxic A-beta Oligomers

Objective: Generate antibodies specific for toxic amyloid-β oligomers(AβO)

Background: Current evidence suggests that propagating prion-likestrains of AβO, as opposed to monomers and fibrils, are preferentiallytoxic to neurons and trigger tau pathology in Alzheimer's disease (AD).In addition, dose-limiting adverse effects have been associated with Aβfibril recognition in clinical trials. These observations suggest thatspecific neutralization of toxic AβOs may be desirable for safety andefficacy.

Design/Methods: Computational simulations were employed as describedherein, using molecular dynamics with standardized force-fields toperturb atomic-level structures of Aβ fibrils deposited in the ProteinData Base. It was hypothesized that weakly-stable regions are likely tobe exposed in nascent protofibrils or oligomers. Clustering analysis,curvature, exposure to solvent, solubility, dihedral angle distribution,and Ramachandran angle distributions were all used to characterize theconformational properties of predicted epitopes, which quantifydifferences in the antigenic profile when presented in the context ofthe oligomer vs the monomer or fibril. The candidate peptide epitopeswere synthesized in a cyclic format that may mimic regional AβOconformation, conjugated to a carrier protein, and used to generatemonoclonal antibodies in mice. Purified antibodies were screened by SPRand immunohistochemistry.

Results:

Sixty-six IgG clones against 5 predicted epitopes were selected forpurification based on their ability to recognize the cognate structuredpeptide and synthetic AβO, with little or no binding to unstructuredpeptide, linker peptide, or Aβ monomers. Additional screening identifiedantibodies that preferentially bound to native soluble AβO in CSF andbrain extracts of AD patients compared to controls. Immunohistochemicalanalysis of AD brain allowed for selection of antibody clones that donot react with plaque.

Conclusion: Computationally identified AβO epitopes allowed for thegeneration of antibodies with the desired target profile of selectivebinding to native AD AβOs with no significant cross-reactivity tomonomers or fibrils.

Example 12

Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptide can be tested in a rat primary cortical neuron assay.

Antibody and control IgG are each adjusted to a concentration such as 2mg/mL. Various molar ratios of A-beta oligomer and antibody are testedalong with a vehicle control, A-beta oligomer alone and a positivecontrol such as the neuroprotective peptide humanin (HNG).

An exemplary set up is shown in Table 9.

Following preincubation for 10 minutes at room temperature, the volumeis adjusted to 840 microlitres with culture medium. The solution isincubated for 5 min at 37 C. The solution is then added directly to theprimary cortical neurons and cells are incubated for 24 h. Cellviability can be determined using the MTT assay.

TABLE 9 AβO/AB AβO AβO AB AB Medium Final volume molar ratio (μL) (μM)(μM) (μL) (μL) (μL) 5/1 1.68 4.2 0.84 12.73 185.6 200 1/1 1.68 4.2 4.2063.64 134.7 200 1/2 1.68 4.2 8.4 127.27 71.1 200 AβO working solution:2.2 mg/mL-500 μM CTRL vehicle: 1.68 μL of oligomer buffer + 127.3 μLPBS + 711 μL culture medium CTRL AβO: 1.68 μL of AβO + 127.3 μL PBS +711 μL culture medium CTRL HNG: 1.68 μL of AβO + 8.4 μL HNG (100 nMfinal) + 127.3 μL PBS + 702.6 μL culture medium

This test was conducted using other antibodies raised against othercyclopeptides comprising other epitopes predicted by the collectivecoordinates method described in Example 1.

Inhibition of A-beta oligomer toxicity was observed for these otherepitopes. Antibodies raised against cyclo(CGHDSGG) (SEQ ID NO: 2) willbe tested.

Example 13

In Vivo Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptide can be tested in vivo in mouse behavioral assays.

The antibody and an isotype control are each pre-mixed with A-beta42oligomers at 2 or more different molar ratios prior tointracerebroventricular (ICV) injection into mice. Control groupsinclude mice injected with vehicle alone, oligomers alone, antibodyalone, and a positive control such as the neuroprotective peptidehumanin. Alternatively, the antibodies can be administered systemicallyprior to, during, and/or after ICV injection of the oligomers. Startingapproximately 4-7 days post ICV injection of oligomers, cognition isassessed in behavioral assays of learning and memory such as the mousespatial recognition test (SRT), Y-Maze assay, Morris water maze modeland novel object recognition model (NOR).

The mouse spatial recognition test (SRT) assesses topographical memory,a measure of hippocampal function (SynAging). The model uses atwo-chamber apparatus, in which the chambers differ in shape, patternand color (i.e. topographical difference). The chambers are connected bya clear Plexiglass corridor. Individual mice are first placed in theapparatus for a 5 min exploration phase where access to only one of thechambers is allowed. Mice are then returned to their home cage for 30min and are placed back in the apparatus for a 5 min “choice” phaseduring which they have access to both chambers. Mice with normalcognitive function remember the previously explored chamber and spendmore time in the novel chamber. A discrimination index (DI) iscalculated as follows: DI=(TN−TF)/(TN+TF), in which TN is the amount oftime spent in the novel chamber and TF is the amount of time spent inthe familiar chamber. Toxic A-beta oligomers cause a decrease in DIwhich can be partially rescued by the humanin positive control.Performance of this assay at different time points post ICV injectioncan be used to evaluate the potential of antibodies raised to thecyclopeptide to inhibit A-beta oligomer toxicity in vivo.

The Y-maze assay (SynAging) is a test of spatial working memory which ismainly mediated by the prefrontal cortex (working memory) and thehippocampus (spatial component). Mice are placed in a Y-shaped mazewhere they can explore 2 arms. Mice with intact short-term memory willalternate between the 2 arms in successive trials. Mice injected ICVwith toxic A-beta oligomers are cognitively impaired and show randombehavior with alternation close to a random value of 50% (versus ˜70% innormal animals). This impairment is partially or completely reversed bythe cholinesterase inhibitor donepezil (Aricept) or humanin,respectively. This assay provides another in vivo assessment of theprotective activity of test antibodies against A-beta oligomer toxicity.

The Morris water maze is another widely accepted cognition model,investigating spatial learning and long-term topographical memory,largely dependent on hippocampal function (SynAging). Mice are trainedto find a platform hidden under an opaque water surface in multipletrials. Their learning performance in recalling the platform location isbased on visual clues and video recorded. Their learning speed, which isthe steadily reduced time from their release into the water untilfinding the platform, is measured over multiple days. Cognitively normalmice require less and less time to find the platform on successive days(learning). For analyzing long-term memory, the test is repeatedmultiple days after training: the platform is taken away and the numberof crossings over the former platform location, or the time of the firstcrossing, are used as measures to evaluate long-term memory. Miceinjected ICV with toxic A-beta oligomers show deficits in both learningand long-term memory and provide a model for evaluating the protectiveactivity of test antibodies.

The Novel Object Recognition (NOR) model utilizes the normal behavior ofrodents to investigate novel objects for a significantly longer timethan known objects, largely dependent on perirhinal cortex function(SynAging). Mice or rats are allowed to explore two identical objects inthe acquisition trial. Following a short inter-trial interval, one ofthe objects is replaced by a novel object. The animals are returned tothe arena and the time spent actively exploring each object is recorded.Normal rodents recall the familiar object and will spend significantlymore time exploring the novel object. In contrast, A-betaoligomer-treated rodents exhibit clear cognitive impairment and willspend a similar amount of time investigating both the ‘familiar’ and‘novel’ object. This can be transiently reversed with known clinicalcognitive enhancers (e.g. donepezil). The NOR assay can be performedmultiple times in longitudinal studies to assess the potential cognitivebenefit of test antibodies.

In addition to behavioral assays, brain tissue can be collected andanalyzed for levels of synaptic markers (PSD95, SNAP25, synaptophysin)and inflammation markers (IL-1-beta). Mice are sacrificed at ˜14 dayspost-ICV injection of oligomers and perfused with saline. Hippocampi arecollected, snap frozen and stored at −80° C. until analyzed. Proteinconcentrations of homogenized samples are determined by BCA.Concentration of synaptic markers are determined using ELISA kits(Cloud-Clone Corp, USA). Typically, synaptic markers are reduced by25-30% in mice injected with A-beta oligomers and restored to 90-100% bythe humanin positive control. Concentrations of the IL-1-betainflammatory markers are increased approximately 3-fold in mice injectedwith A-beta oligomers and this increase is largely prevented by humanin.These assays provide another measure of the protective activity of testantibodies at the molecular level.

Example 14

In Vivo Propagation Inhibition Assay

In vivo propagation of A-Beta toxic oligomers and associated pathologycan be studied in various rodent models of Alzheimer's disease (AD). Forexample, mice transgenic for human APP (e.g. APP23 mice) or human APPand PSEN1 (APPPS1 mice) express elevated levels of A-beta and exhibitgradual amyloid deposition with age accompanied by inflammation andneuronal damage. Intracerebral inoculation of oligomer-containing brainextracts can significantly accelerate this process (13, 14). Thesemodels provide a system to study inhibition of A-beta oligomerpropagation by test antibodies administered intracerebrally orsystemically.

Example 15

CDR Sequencing

Clone 25 (303-25) which was determined to have an IgG2A heavy chain anda kappa light chain was selected for CDR and variable regions of theheavy and light chains.

RT-PCR was carried out using 5′ RACE and gene specific reverse primerswhich amplify the appropriate mouse immunoglobulin heavy chain(IgG1/IgG3/IgG2A) and light chain (kappa) variable region sequences.

The specific bands were excised and cloned into pCR-Blunt II-TOPO vectorfor sequencing, and the constructs were transformed into E. coli

At least 8 colonies of each chain were picked & PCR screened for thepresence of amplified regions prior to sequencing. Selected PCR positiveclones were sequenced.

The CDR sequences are in Table 10. The consensus DNA sequence andprotein sequences of the variable portion of the heavy and light chainare provided in Table 11

TABLE 10 Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GYTFTSYW 17 CDR-H2IDPSDSQT 18 CDR-H3 SRGGY 19 Light CDR-L1 QDINNY 20 CDR-L2 YTS 21 CDR-L3LQYDNLWT 22

TABLE 11Consensus DNA sequence and translated protein sequences of the variable region. Thecomplementarity determining regions (CDRs) are underlined according to IMTG/LIGM-DB.Isotype Consensus DNA Sequence Protein sequence IgG2aATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA MGWSCIILFLVATATG SEQ IDGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTG VHSQVQLQQPGAELVRNO: 23, 24 GTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCT GGC PGASVKLSCKASGYTF TACACCTTCACCAGCTACTGG ATGAACTGGGTGAAGCAGAGGCCT TSYW MNWVKQRPGQGLGGACAAGGCCTTGAATGGATTGGTATG ATTGATCCTTCAGACAGT EWIGM IDPSDSQT HYN CAAACTCACTACAATCAAATGTTCAAGGACAAGGCCACATTGACT QMFKDKATLTVDKSSSGTAGACAAATCCTCCAGCACAGCCTACCTGCAGCTCAGCAGCCTG TAYLQLSSLTSEDSAVACATCTGAGGACTCTGCGGTCTATTACTGT TCAAGAGGGGGCTAC YYC SRGGY WGQGTTLTTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA VSS KappaATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTT MRPSIQFLGLLLFWLH SEQ IDCATGGTGCTCAGTGTGACATCCAGATGACACAGTCTCCATCCTCA GAQCDIQMTQSPSSLSNO: 25, 26 CTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCA ASLGGKVTITCKASQD AGC CAAGACATTAACAACTAT ATAGCTTGGTACCAACACAAGCCT INNY IAWYQHKPGKGPGGAAAAGGTCCTAGGCAGCTCATATAT TACACATCT ACATTGCAG RQLIY YTS TLQPGIPSCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGAT RFSGSGSGRDYSFTISTATTCCTTCACCATCAGCGACCTGGAGCCTGAAGATATTGCAACT DLEPEDIATYYC LQYDTATTATTGT CTACAGTATGATAATCTGTGGACG TTCGGTGGAGGC NLWT FGGGTKLEIKACCAAGCTGGAAATCAAA

TABLE 12 A-beta “Epitope” Sequences and select A-betasequences with linker HDSG (SEQ ID NO: 1)CGHDSGG, cyclo(CGHDSGG) (SEQ ID NO: 2) HDSGY (SEQ ID NO: 4)RHDSG (SEQ ID NO: 5) RHDS (SEQ ID NO: 6) GHDSG (SEQ ID NO: 7)GHDSGG (SEQ ID NO: 8) GGHDSGG (SEQ ID NO: 9) GHDSGGG (SEQ ID NO: 10)HDSGYE (SEQ ID NO: 11) CGHDSGGC (SEQ ID NO: 12) RHDSGY (SEQ ID NO: 13)DSGY (SEQ ID NO: 14) DSGYEV (SEQ ID NO: 15) FRHDSG (SEQ ID NO: 16)Cyclo(CGHDSG-PEG2); CGHDSG-PEG2(SEQ ID NO: 27)Cyclo(C-PEG2-HDSGG); C-PEG2-HDSGG (SEQ ID NO: 28)

TABLE 13 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3)

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

The scope of the claims should not be limited by the preferredembodiments and examples, but should be given the broadestinterpretation consistent with the description as a whole.

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

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The invention claimed is:
 1. An isolated antibody that specificallybinds to an amyloid-beta (A-beta) peptide having a sequence of HDSG (SEQID NO:1) or a related epitope sequence in A-beta the antibody comprisinga light chain variable region and a heavy chain variable region, theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, the light chain variable regioncomprising complementarity determining regions CDR-L1, CDR-L2 and CDR-L3and with the amino acid sequences of said CDRs comprising the sequences:(SEQ ID NO: 17) CDR-H1 GYTFTSYW (SEQ ID NO: 18) CDR-H2 IDPSDSQT(SEQ ID NO: 19) CDR-H3 SRGGY (SEQ ID NO: 20) CDR-L1 QDINNY(SEQ ID NO: 21) CDR-L2 YTS (SEQ ID NO: 22) CDR-L3 LQYDNLWT.


2. The antibody of claim 1, wherein the antibody is a conformationspecific and/or selective antibody that specifically or selectivelybinds to HDSG (SEQ ID NO:1) or a related epitope peptide presented in acyclic compound, preferably a cyclic peptide having a sequence as setforth in SEQ ID NO: 2, 12, 28 or
 29. 3. The antibody of claim 1, whereinthe antibody is a monoclonal antibody and/or wherein the antibody is ahumanized antibody.
 4. The antibody of claim 1, wherein the antibody isan antibody binding fragment selected from Fab, Fab′, F(ab′)2, scFv,dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and multimersthereof.
 5. The antibody of claim 1, wherein the light chain variableregion and the heavy chain variable region are fused.
 6. The antibody ofclaim 1, wherein the heavy chain variable region comprises: A. i) anamino acid sequence as set forth in SEQ ID NO: 24; ii) an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80%, orat least 90% sequence identity to SEQ ID NO: 24, wherein the CDRsequences are as set forth in SEQ ID NO: 17, 18 and 19, or iii) aconservatively substituted amino acid sequence of A. i), and/or whereinthe antibody comprises a light chain variable region comprising: B. i)an amino acid sequence as set forth in SEQ ID NO: 26, ii) an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80%, orat least 90% sequence identity to SEQ ID NO: 26, wherein the CDRsequences are as set forth in SEQ ID NO: 20, 21 and 22, or iii) aconservatively substituted amino acid sequence of B. i).
 7. The isolatedantibody of claim 6, wherein the heavy chain variable region amino acidsequence is encoded by a nucleotide sequence as set forth in SEQ ID NO:23 or a codon degenerate or optimized version thereof; and/or theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 25 or a codondegenerate or optimized version thereof.
 8. The isolated antibody ofclaim 2, wherein the antibody selectively binds A-beta oligomer overA-beta monomer and/or A-beta fibril.
 9. The isolated antibody of claim8, wherein the antibody is at least 2 fold and more selective for A-betaoligomer over A-beta monomer and/or A-beta fibril.
 10. Animmunoconjugate comprising the antibody of claim 1 and a detectablelabel or cytotoxic agent, optionally wherein the detectable labelcomprises a positron emitting radionuclide.
 11. A composition comprisingthe antibody of claim 1, or an immunoconjugate comprising said antibody,optionally with a diluent.
 12. A cell expressing an antibody of claim 1,optionally wherein the cell is a hybridoma.
 13. A kit comprising theantibody of claim 1 or an immunoconjugate comprising said antibody or acell expressing said antibody.
 14. A method of determining if abiological sample comprises A-beta, the method comprising: a. contactingthe biological sample with an antibody of claim 1 or an immunoconjugatecomprising said antibody; and b. detecting the presence of any antibodycomplex.
 15. A method of measuring a level of A-beta in a subject, themethod comprising: administering to a subject at risk of, suspected ofhaving, or having Alzheimer's Disease (AD), an immunoconjugatecomprising the antibody of claim 1, wherein the antibody is conjugatedto a detectable label; and detecting the label, optionally wherein thelabel is a positron emitting radionuclide.
 16. A method of inhibitingA-beta oligomer propagation, the method comprising contacting a cell ortissue expressing A-beta with an effective amount of the A-beta specificantibody of claim 1, or an immunoconjugate comprising said antibody, toinhibit A-beta aggregation and/or oligomer propagation.
 17. A method oftreating AD or other disease characterized by A-beta amyloid pathology,the method comprising administering to a subject in need thereof aneffective amount of the antibody of claim 1 or a pharmaceuticalcomposition comprising said antibody.